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Table of Contents   
ORIGINAL ARTICLE  
Year : 2017  |  Volume : 10  |  Issue : 6  |  Page : 1518-1523
Evaluation of oxidative stress marker in patients with human papillomavirus infection


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran,, Iran
2 Nephrology and Kidney Transplant Research Center, Urmia University of Medical Sciences, Urmia, Iran
3 Department of Biochemistry, Faculty Member of Islamic Azad University, Science and Research Branch of Tehran, Tehran, Iran
4 Solid Tumor Research Center, Urmia University of Medical Sciences, Urmia, Iran
5 Department of Biostatistics and Epidemiology, Patient Safety Research Center, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran

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Date of Web Publication11-Jan-2018
 

   Abstract 


Introduction and Objective: Human papillomavirus(HPV) unenveloped viruses with double-stranded circular DNA cause infection in skin epithelial cells and mucosal surfaces. HPV infection has been detected in more than 70% of cervical cancers throughout the world. Among the effective factors in the progression of HPV infection are oxidative stress and oxidative markers, and the oxidative stress during viral infection can be due to the host immune responses to viral proteins or as a result of viral genes expression. This study aims to compare the mean serum level of oxidative malondialdehyde(MDA) marker and the mean of 8-Hydroxyguanosine(8- OhdG) in two groups of healthy people and patients with HPV infection. Methods: This is a descriptive, cross-sectional study in which 30patients with HPV infection and 35 healthy people(as the control group) were selected using convenience sampling method. After obtaining written consent from the subjects, the researchers took fasting blood samples and morning urine samples from them. Serum MDA was measured using the spectrophotometric method, and 8-OHdG was measured using the competitive enzyme-linked immunosorbent assay method. Finally, the data were analyzed using SPSS version 17. Results: The results of this study showed that the mean and standard deviation of serum MDA concentration have increased significantly in patients with HPVs infection(4.56±1.64 nM) than in the control group(1.64±0.37 nM). The mean and standard deviation of urinary concentration(8-OHDG) in patients with HPV infection was 14.61±1.39ng/ml while it was 9.66±1.74ng/ml in the control group. It was significantly higher in people who had high-risk HPV infection than in the group with low-risk HPV(P<0.001). Conclusion: Based on the results of this study, oxidative reactions and biological damage caused by these reactions as a result of the increased oxidative stress in the body can be suggested as a mechanism involved in the progression of the cervical papillomavirus infection and the cancer caused by it.

Keywords: 8-hydroxydeoxyguanosine, human papillomavirus, malondialdehyde, oxidative stress

How to cite this article:
Hamzyan Olia JB, Khadem Ansari MH, Yaghmaei P, Ayatollahi H, Khalkhali HR. Evaluation of oxidative stress marker in patients with human papillomavirus infection. Ann Trop Med Public Health 2017;10:1518-23

How to cite this URL:
Hamzyan Olia JB, Khadem Ansari MH, Yaghmaei P, Ayatollahi H, Khalkhali HR. Evaluation of oxidative stress marker in patients with human papillomavirus infection. Ann Trop Med Public Health [serial online] 2017 [cited 2018 May 21];10:1518-23. Available from: http://www.atmph.org/text.asp?2017/10/6/1518/222660



   Introduction Top


Human papillomavirus(HPV) infection is one of the most common sexually transmitted infections detected in more than 70% of cervical cancers throughout the world, which is the second cause of death among the malignant diseases that females face in the world.[1] The World Health Organization has identified 12 types of HPVs(16, 18, 31, 33, 35, 39, 45, 51, 56, 52, 58, and 59) as high risk or carcinogenic types and some other HPVs(68, 73, 26, 30, 53, 66, 67, 69, 82 and 85) as a type that may be carcinogenic.[2] Of these, HPV(HPV16) and HPV18 have been detected in about 71% of cervical cancer cases throughout the world.[3] Other types of HPV genotypes(6, 11, 13, 40, 42, 43, 44, 54, 61, 70, 72, 81, and 89), mostly associated with genital warts, are classified as low-risk types. The prevalence of HPV in different regions, countries, and even in different demographic subpopulations within a single country has been shown to be different. However, HPV has the global prevalence of 7/11%.[4],[5]

Several factors are effective in the HPV infection(including the age of onset of sexual activity, the number of sexual partners, the economic status, smoking, the number of pregnancies).[6],[7] Other factors that contribute to the progression of the HPV infection are the viral oxidative stress and the levels of oxidant markers. Oxidative stress is associated with several chronic diseases in terms of etiology.[8],[9] Many of the biological goals of oxidative stress are lipids as a type of biomolecules involved. Lipid oxidation leads to the production of a number of secondary products, which are mainly aldehyde products with high-oxidative damage ability. The long life and high reactivity of these molecules to internal and external cells and their reactions to biomolecules such as nucleic acids and proteins have caused irreversible damages to cell function.[10],[11] Malondialdehyde(MDA) is the most important studied product as a result of peroxidation of unsaturated fatty acids. Since the 1960s, various methods have been developed to evaluate this molecule to quantify the levels of oxidative stress in invivo and invitro conditions.[12] Oxidative cell damage is an identified mechanism in cell and tissue damage caused by free radicals and reactive oxygen species(ROS).[13] ROS, which are produced by mitochondrial respiration metabolism, are normally neutralized by antioxidant defense mechanisms, but when free radicals' production predominates over enzymatic and nonenzymatic antioxidant defense, oxidative stress is the result, which plays a significant role in the pathogenesis of many diseases.[14] Following the oxidative stress, the lipid peroxidation level is also increased. Lipid peroxidation is a chain reaction that paves the way for the continuity of peroxidation through the continuous production of free radicals. Free radicals can connect with cell components, react to the unsaturated bonds of membrane lipids, destroy proteins, and attack nucleic acids.[15] On the other hand, ROS leads to the production of MDA, which disables the membrane carrier, thereby applying its own carcinogenic effect.[16] MDA is considered to be the most important biochemical marker for determining the lipid peroxidation. In this process, free radicals damage the saturated fatty acids, which are in fact the main components of the cell membrane, and lead to the formation of MDA following the lipid peroxidation. In addition, free radicals can produce MDA by directly affecting the DNA and its oxidation.[11] Among the purine and pyrimidine bases, guanine is more capable of oxidation, so that a compound called 8-Hydroxyguanosine(8-OhdG) is produced as a result of radical hydroxyl attack to the eighth position of the guanine molecule.[4] This compound is released as a result of DNA repair processes and is urinated without metabolism. The mutagenic property of 8-OHdG is quite known.[5] Therefore, this compound is considered as an important oxidative cell biomarker and DNA repair product.[6] Since 8-OHdG shows the dynamic balance between the oxidative damage of DNA and its repair speed, the measurement of this compound is important in the DNA damage measurement in the entire body.[7],[6]

8-hydroxydeoxyguanosine is one of the biological markers of cell damage.[17] This mutagenic compound is produced as a result of DNA repair processes and is considered as an important oxidative stress factor and DNA repair product. Considering that this compound shows the dynamic equilibrium between the oxidative damage of DNA and the repairing speed, the measurement of this compound is important in DNA damage measurement.[18],[19] Many studies have shown the increased lipid peroxidation in HPV infection. However, it is not yet clear whether or not lipid peroxidation changes in people with HPV infection, depending on the type of HPV. On the other hand, whether the level of oxidative damage to DNA is associated with an increase in the infection-induced damages the markers of which are the high-risk HPV types have not been studied completely yet. Therefore, this study has been conducted with the aim of determining the damage levels of oxidative DNA and lipids and its association with HPV infection and its resultant cancer in women with HPV infection.


   Methods Top


This cross-sectional study was conducted using the convenience sampling method in Urmia during a 1-year period from December 2015 to 2016. Atotal of 30patients with HPVs who had been admitted to the Specialty Department of Women at Shahid Motahari Hospital of Urmia, and a total of 35 healthy persons who were consistent with the patients in terms of age and sex(women with sexual activity, between 25 and 45years of age) were selected for study. The inclusion criteria of the study were being between 25 and 45years old and having sexual activity. People who took antioxidant drugs and fat-lowering drugs and had a history of kidney, liver, diabetes, or cigarette smoking were excluded from the study. Fasting blood samples and morning urine samples were taken from all of the subjects. The blood samples were stored for 15min in a laboratory, and the serum was separated using a benchtop centrifuge and the samples were stored in a- 70°C freezer until the experiment time. The MDA levels of serum samples were measured spectrophotometry in the Biochemistry Laboratory of Urmia University of Medical Sciences.

Measuring the malondialdehyde level

To measure the level of MDA as a lipid peroxidation marker, we used the Nalondi™ Lipid Peroxidation Assay Kit(NID) made by the Navand Salamat Company in Iran. The Nalondi™ Lipid Peroxidation Assay Kit is repeatable and provides a standard for measuring the MDA level and knowledge of lipid peroxidation in biological samples such as serum, plasma, urine, homogeneous tissue, cell lysate, and culture medium liquid. In summary, MDA reacts to Thiobarbituric acid at high temperatures and produces a pink product that is measured by colorimetric method at the wavelength of 540–530nm.

Measuring the 8-hydroxyguanosine(urinary oxidative stress marker) level

The urinary concentration of 8-hydroxyguanosinee was measured by Human 8-OHDG enzyme-linked immunosorbent assay Kit made by China's Technology Laboratory. To measure the 8-OHDG level, the fasting morning urine samples were placed in sterile containers, centrifuged for 2–3min at 2000–3000 RPM, poured in sterile Eppendorf tubes and stored at-20°C until the experiment time. During the experiment time, the urine specimen were incubated with the primary antibody solution in wells previously coated with 8-OHdG at 37°C for 1 h. At this time, the antibody works competitively to connect with 8-OHdG in the sample and well. The conjugated antibodies were washed. The second antibody was added to the environment and conjugated to antibodies already conjugated to 8-OHdG in the well. The nonconjugated secondary antibodies were also washed. Then, chromatography and diluent solution were added and incubated for 10min at 37°C. Finally, the reaction solution was added and examined at the wavelength of 450nm. The values of this compound were obtained using the standard curve in ng/ml.

Finding

The results of this study were analyzed usingSPSS version17 (Chicago: SPSS Inc.). The one-sample Kolmogorov–Smirnov test showed that the (MDA serum concentration) measurement does not follow a normal distribution(P<0.001). The mean and standard deviation of serum MDA concentration was(56.4±1.64 nM) in patients with HPV infection and was(1.64±0.37 nM) in the control group. The Mann-Whitney U test showed a significant difference(P<0.001). [Table1] summarizes the results of the descriptive statistics of MDA(serum MDA concentration) measurement in the group of patients with papillomavirus infection and the control group.
Table 1: Descriptive statistics markers of malondialdehyde (serum malondialdehyde concentration) measurement in the control group and the group of patients with papillomavirus infection

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The mean and standard deviation of serum MDA concentration were(6.55±0.58nm) in the group with high-risk HPV while they were(3.85±0.66 nM) in the group with low-risk HPV and were(5.76±0.97) in the coinfection group. The results of the Kruskal–Wallis test showed a significant difference between the mean concentration of MDA in the three groups(P<0.001). The mean serum MDA concentration level was lower in patients with HPV infection than in the two other groups. [Table2] summarizes the results of the descriptive statistics of MDA(serum MDA concentration) measurement in the group of patients with high/low-risk HPV infection and the co-infection group.
Table 2: Descriptive statistics markers of malondialdehyde (serum malondialdehyde concentration) measurement in groups of patients with high/low-risk human papillomavirus infection and the coinfection group

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The one-Sample Kolmogorov–Smirnov test showed that the measurement of urinary 8-hydroxyguanosine concentration does not follow a normal distribution(P=0.031). The mean and standard deviation were 14.61±1.39ng/ml in patients with HPV infection while they were 9.66±1.74ng/ml, which the Mann-Whitney U test showed a statistically significant difference(P<0.05, P<0.001)[Table3].
Table 3: Summarizes the results of the descriptive statistics of 8-hydroxydeoxyguanosine in the group of patients with human papillomavirus infection and the control group

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   Discussion Top


MDA and 8-hydroxyguanosinee are regarded as the most important markers of oxidative stress. MDAis a three-carbon compound formed through the peroxidation of saturated fatty acids, especially arachidonic acid. MDAis one of the final products of membrane lipid peroxidation. Since the level of MDAin various diseases increases with the increase of free oxygen radicals, a relationship has been observed between free radicals and damages.[14] According to a review study conducted by Rahal et al., the world today is exposed to increased chronic diseases such as cardiovascular disorders, cancer, etc., and the negative effects of these diseases on health and mortality. Moreover, the findings of this study show that oxidative stress is nothing but an imbalance between oxidants and antioxidants in favor of oxidants, which are formed as a natural product of aerobic metabolism, but the amount of these products increases during these pathophysiological conditions.[13]

A study carried out by Siegel et al. showed that women with HPV infection and high-oxidant markers had a greater chance of getting rid of infection than those with low oxidation markers. This association is limited to the oncogenic type of HPV and possibly HPV16 infection. This study also found that MDA increased as an oxidant marker as a result of infection and immune response to infection. The high levels of MDA biomarker indicate infection, especially the oncogenic types of HPV. These findings reflect the association between oxidant biomarkers and immune response to HPV infection. These findings showed that ROS plays a role in the HPV immune response, and oxidation markers may indicate an active and ongoing immune response to HPV infection.[20]

According to the findings ofThanan etal.(2015) about the role of oxidative stress in neurodegenerative diseases and cancer, 8-hydroxyguanosine can be a strong biomarker. Therefore, the production of ROS occurs during chronic carcinogenesis resulting from bacterial and viral infections, as a result of DNA damage in infected tissues, which leads to the progression of progressive tissue inflammation and the increased risk of cancer.[14]

A comparative study conducted by Taghiyar and Razmi on the serum levels of MDAin patients with breast cancer and healthy people in Isfahan showed that measuring the level of MDA in plasma or serum as a marker of lipid peroxidation and an appropriate noninvasive biomarker for measuring oxidative stress, which is often used in adults for the estimation of the physiological or pathological conditions created by free radicals. It can thus be said that the decreased level of antioxidants and the increased level of oxidative stress in the body that results in lipid peroxidation and eventually MDA production are clearly associated with an increased risk of breast cancer. It seems that MDA can be suggested as an auxiliary marker of the impact of breast cancer on the continuation of treatment.[21] The results of a study performed byMazdak etal.(2009)on the evaluation of oxidative stress markers in bladder cancer patients in comparison with the control group indicate that peroxidation lipid is a process involved in bladder cancer and MDA can be considered as an auxiliary marker of bladder cancer. In addition, several studies have been conducted on the change of this marker following the treatment, and therefore, the possibility of using it as a noninvasive marker in determining the success rate of treatment.[22] A study carried out by Williams et al. on the induction of oxidative stress, and DNA damage by HPV infection type16 revealed that oxidative stress is produced during a commonly occurring viral infection and inflammatory response to infection, resulting in the release of ROS from neutrophils and macrophages. This study also showed that the expression of E6 HPV 16 protein isoforms increases the oxidative stress level, causing damage to the oxidative genome in host cells.[23] A study conducted by Shiva regarding the effects of HPV infection and inflammation on the incidence of cancer indicated a strong association between cancer and chronic infection related to the production of ROS released during infection. ROS strongly damage the DNA of the cell. They also prevent from the recovery and base-excision in DNA. The increased mitotic effects in response to inflammation puts more cells at the risk of mutation. In addition, apoptosis, which plans the death of damaged cells, ceases in infectious and inflamed tissues.[24]

With the increased level of prostaglandin, the infection increases the production of reactive species of oxygen and nitrogen, which in turn causes the expression of anti-inflammatory cytokines such as Interferon-γ, Tumor necrosis factor-α, Interleukin-6(lL-6) and lL-1. These anti-inflammatory cytokines are responsible for increasing the production of free radicals through protein-kinase-associated signaling pathways. Therefore, the ROS and RON produced by this method can react to a variety of macromolecules such as DNA in mitotic cells so that permanent gene mutations such as point mutations, gene removal, and gene reconstruction can occur. Under normal conditions, cells usually reduce the levels of ROS and RON by activating the antioxidant systems. They also begin to repair the damaged by activating the genes responsible for DNA repair. However, this DNA damage is accumulated and not repaired in chronic infection, and as a result, chronic infection and inflammation causes oxidative stress, which has deleterious effects on the cells. Free radicals react with all the components of the cell to form sustained excess compounds. The ROS-induced oxidative DNA damage includes single-stranded or double-stranded DNA breaks, open DNA changes, additional interstrand DNA compounds, and cross-links to DNA proteins.[25]


   Results Top


This study showed a significant increase in the urinary levels of 8-Hydroxyguanosine and serum MDA levels in patients with high-risk HPV in comparison with those with a low risk of HPV infection. The lower levels of 8-OHdG and MDA in the control group than in the patient group indicates an increased risk of mutagenesis in these patients, resulting in a cancerous infection of the HPV group of the high-risk type. Finally, considering the significant positive association between infection with the HPV virus and urinary excretion of 8-OHdG, we can conclude that using proper screening methods, preventing the infection and training the patients with HPV infection can help reduce the oxidative damage. Moreover, the increased levels of MDA in patients with HPV indicated that continuous infection and suffering from the high-risk genotypes of HPV may increase the production of free radicals, which is effective in the lipid peroxidation.


   Conclusion Top


The final result is that the oxidative reactions and biological damages caused by these reactions as a result of the increased oxidative stress in the body can be suggested as a mechanism involved in the progression of the cervical infection and the creation of cancer induced by it.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Mohamad Hasan Khadem Ansari
Nephrology and Kidney Transplant Research Center, Urmia University of Medical Sciences, Urmia
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ATMPH.ATMPH_464_17

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