Oxidative stress and antioxidant modulation in cervical infection by the human papillomavirus

Oxidative stress and antioxidant modulation in cervical infection by the human papillomavirus | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 May 2025 V1 Latest version Share on Oxidative stress and antioxidant modulation in cervical infection by the human papillomavirus Authors : Tatiana Mugnol Schöffel 0000-0002-1003-5800 [email protected] , Lara Kochenborger , Taiana Pedrotti , Nathália Billig Garces , Vanessa Jung Ferreira Flores , and Janaina Coser Authors Info & Affiliations https://doi.org/10.22541/au.174679804.45741205/v1 Published Cadernos Cajuína Version of record Peer review timeline 254 views 121 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Backgrounds: Human papillomavirus (HPV) infection is the main risk factor for cervical cancer. However, other cofactors are involved in the development of cervical carcinogenesis, such as oxidative stress, and can contribute to the persistence of HPV infection in the epithelium and to its integration into the cell genome. The objective of this study was to assess markers of oxidative stress in cervical samples from women infected with cervical HPV, with or without cytological abnormalities. Methods: Cytopathological exams, molecular detection of HPV, and markers of oxidative stress of cell samples from the cervix of 35 women were assessed. Results : The HPV-B group exhibited higher levels of TBARS, GSH, GST, and CP compared to the CG and HPV-A groups, while CAT levels were similar across all groups. CP concentrations were lower in the HPV-A group. Pearson’s correlation analysis showed a positive correlation between CP and GST in the CG group, CP and TBARS in the HPV-A group, and GSH and TBARS in the HPV-B group, highlighting distinct oxidative stress profiles among the groups. Conclusions: The results revealed that women infected by HPV with abnormal cervical cells exhibited higher lipid peroxidation and protein oxidation, although an antioxidant response was also observed. Introduction Cervical cancer is the fourth-most-common neoplasm among women [1]. The disease is preceded by precursor lesions, and infection by the human papillomavirus (HPV) is a known risk factor for developing this cancer [2]. HPV infection is the most prevalent sexually transmitted infection (STI) worldwide, and most sexually active individuals acquire it at some point in their lives [3]. These infections are transient in most cases, produce no signs or symptoms, and are eliminated spontaneously by immune system action. However, some infections become persistent and increase the risk of pre-neoplastic lesions that can progress to neoplasms [4]. Thus, although HPV infection is a necessary component inducing cervical carcinogenesis, the infection alone is insufficient to cause the cancer. Other risk factors include immunosuppression, tobacco use, and sexual and reproductive factors, such as early sexual debut, numerous partners and history of STI [5]. Oxidative stress has also been identified as a cofactor in cervical carcinogenesis by contributing to persistence of HPV infection in the epithelium and to its viral integration into the cell genome [6-8]. These events are essential to HPV-mediated carcinogenesis, resulting in cellular changes, abnormal proliferation and malignant progression [4]. Oxidative stress is caused by an imbalance between oxidants and antioxidants with excess of the former, leading to over-generation of reactive oxygen species (ROS). These excess ROS promote biological injury to cell structures and reaction with proteins, carbohydrates, nucleic acids and lipids [9]. Therefore, the objective of this study was to assess markers of oxidative stress in cervical samples from women infected with cervical HPV, with or without cytological abnormalities. Materials and methods An observational, cross-sectional study, previously approved by the Research Ethics Committee of the Universidade de Cruz Alta – UNICRUZ under permit no. 2.790.225, was conducted. The study population comprised women who had sexually debuted, were aged 18-65 years, and had visited public or private health services for a Papanicolaou (smear) in the cities of Cruz Alta and Ijuí, Rio Grande do Sul state, Brazil, between September 2018 and June 2021. Women who were pregnant or had undergone hysterectomy were excluded. A non-probabilistic convenience sample comprising 35 women was investigated. For all participants, samples of cells were collected from the cervix, using an endocervical brush and Ayre´s spatula, for cytopathological exam, HPV DNA detection and assessment of markers of oxidative stress. Regarding the cytopathological exams, the conventional method or liquid medium was employed, depending on availability at the health services involved. Slides were examined independently by 2 cytologists and results classified according to the Bethesda System [10]. In the event of disagreement, a third cytologist reviewed the slides. For HPV-DNA testing, samples underwent extraction using the silica-based method [11] and were amplified using the Nested-PCR method [12]. Markers of oxidative stress were measured using spectrophotometric techniques. Lipid peroxidation was determined using the thiobarbituric acid reactive substance (TBARS) assay, as per the adapted protocol of Buege & Aust [13], with measurements performed at 535nm. Carbonylated proteins (CP) were analyzed using the adapted technique of Levine et al. [14], with measurements at 370 nm. Reduced glutathione (GSH) levels were measured according to the adapted method of Elman [15] at 412nm. Glutathione S-transferase (GST) activity was determined using the method of Habig et al . [16], with adaptations, at 340nm. Catalase (CAT) activity was determined using the technique of Nelson & Kiesow [17] with adaptations, and measurements performed at 240nm. Total protein levels were measured using a NanoDrop® 2000 Thermo Scientific spectrophotometer. Based on the results of these analyses, women were stratified according to results of molecular testing and cytopathological exam into the following groups: Control group – CG (n=15): containing women who were negative for HPV on the molecular test and negative for intraepithelial lesion or malignancy on the cytopathological exam, with no detection of any other pathogenic agents ( Candida spp., Gardnerella vaginalis or Trichomonas vaginalis). HPV-A Group (n=16): containing women who were positive for HPV on the molecular test and negative for intraepithelial lesion or malignancy on the cytopathological exam, with no detection of any other pathogenic agents ( Candida spp., Gardnerella vaginalis or Trichomonas vaginalis). HPV-B Group (n=4): containing women who were positive for HPV on the molecular test and exhibited cell changes on the cytological exam. There were 2 cases of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H), and 2 cases of low-grade squamous intraepithelial lesion (LSIL), without the presence of other pathogenic agent identified by cytopathological exam ( Candida spp., Gardnerella vaginalis or Trichomonas vaginalis). Statistical Analysis ANOVA Prior to the analysis of variance, the meeting of assumptions of homogeneity of variances and normality of errors were investigated by applying the Bartlett and Shapiro-Wilk tests, respectively, with a probability of error of 5%. When assumptions were not met, the Box-Cox procedure was used to check the adequate transformation of data with the software Action [18]. Results were submitted to analysis variance and means compared by the Scott-Knott test at a probability of 5%, using the Sisvar 5.6 statistical package [19]. Pearson´s Correlation The linear relationships between the variables TBARS, GSH, GST, CAT and CP for the CG, HPV-A and HPV-B groups were determined using Pearson´s coefficient of correlation with the aid of the Genes software [20]. Results Overall, mean age of the participants was 33.11 years. By group, mean age was 35.13 years in the CG, 32.68 in the HPV-A, and 27.25 years in the HPV-B group. The analysis of variance between the markers of oxidative stress for the CG, HPV-A and HPV-B groups revealed higher levels of TBARS, GSH, GST and CP in the HPV-B group (Table 1). With regard to CAT, similar levels were found across all groups, and likewise there was no group difference for mean age. However, CP concentrations were lower in the HPV-A group. CG 2.18 b 0.02 b 0.08 b 4.81 a 1742.91 b 35.13 a HPV-A 1.89 b 0.03 b 0.08 b 2.64 b 252.86 c 32.69 a HPV-B 8.79 a 0.10 a 0.22 a 3.88 ab 4701.32 a 27.25 a Table 1 . Analysis of variance (ANOVA) between TBARS, GSH, GST, CAT, CP and patient age in CG, HPV-A and HPV-B groups. Thiobarbituric acid reactive substance (TBARS); Reduced Glutathione (GSH); Glutathione-S-Transferase (GST); Catalase (CAT); Carbonylated proteins (CP). Note: TBARS results expressed as nmol/mg protein, GSH as µmol GSH/mg protein, GST as µmol GS-DNB/min/mg protein, CAT as nmol/mg protein/min and CP as nmol of carbonylated protein/mg of total protein. *means not followed by same letter differ on the t -test for independent samples at probability of 5%. ¹transformed data. The analysis of Person´s correlation between the markers of oxidative stress of patients in the CG, HPV-A and HPV-B groups (Table 2) revealed a positive correlation between CP and GST in the CG group. In the HPV-A group, there was a positive correlation between CP and TBARS, whereas in the HPV-B group, a positive correlation was found between GSH and TBARS. TBARS 0.36 0.42 -0.09 0.29 0.36 GSH 0.20 0.01 -0.17 -0.17 GST 0.33 0.79* 0.02 CAT 0.11 -0.33 CP 0.15 AGE HPV-A TBARS GSH GST CAT CP AGE TBARS 0.39 -0.05 0.25 0.75* 0.14 GSH -0.11 0.04 -0.13 0.42 GST 0.04 -0.13 0.23 CAT 0.21 -0.37 CP -0.06 AGE HPV-B TBARS GSH GST CAT CP AGE TBARS 0.97* -0.01 -0.77 0.44 -0.68 GSH 0.07 -0.71 0.54 -0.48 GST -0.51 -0.55 0.19 CAT 0.21 0.69 CP 0.11 AGE Table 2 . Pearson´s coefficient of correlation between TBARS, GSH, GST, CAT, CP and patient age in CG, HPV-A and HPV-B groups. Thiobarbituric acid reactive substance (TBARS); Reduced Glutathione (GSH); Glutathione-S-Transferase (GST); Catalase (CAT); and Carbonylated proteins (CP). * Significant coefficients on t -test at probability of 5%. Discussion In the present study, the markers of oxidative stress TBARS, CP, GSH, GST and CAT were assessed in cells exfoliated from the cervix. The results revealed that women infected by HPV with abnormal cervical cells exhibited a higher concentration of TBARS, indicating membrane peroxidation in the presence of infection by the virus and of cell abnormalities [21, 22]. These findings corroborate evidence in the literature showing higher levels of TBARS in blood samples of HPV-infected women and also of those with squamous intraepithelial lesion (SIL) and genital warts [21, 23, 24]. Elevated TBARS concentration may be associated with HPV-induced oxidative stress through reactive species imbalance. In addition, viral infection and the establishment of persistence of the infection are exacerbated by reactive species [25]. Protein oxidation was also found to be high in this group, induced directly by reactive species or indirectly by reactions with secondary products of oxidative stress, where CP serve as a biomarker of severe oxidative damage of proteins [26]. However, CP levels were lower in women exhibiting HPV infection with no cell abnormalities. Women infected by HPV that also exhibited cell changes had higher levels of GSH and GST activity, indicating antioxidant response to suppress the oxidative stress induced by HPV and resultant lesions [21, 24-28]. GSH is part of total GSH and constitutes an important cell antioxidant, helping to combat damage from lipid peroxidation and membrane damage, while protecting against ROS [29-31]. Increased GST activity is important for restoring antioxidant levels and stimulating enzymes to neutralize the deleterious effects of ROS [32]. Additionally, the assessment of markers of oxidative stress provides a measure of oxidative stress level and possible modulations. Women uninfected by HPV and without cell abnormalities showed a positive correlation between CP and GST where, under normal conditions, redox homeostasis is maintained by the pro and antioxidant systems [9, 33]. Individuals infected by HPV but without cell abnormalities showed a positive correlation between CP and TBARS, bearing in mind that elevated ROS results in damage to cell structures and impairs their functioning, favoring carcinogenesis [9, 34]. Women infected by HPV and exhibiting cell abnormalities showed a positive correlation between TBARS and GSH. These findings suggest an antioxidant response to lipid peroxidation, given that GSH acts in combatting damage from lipid peroxidation [35, 36]. The study has some limitations, such as the small sample size. Nevertheless, the results help elucidate the role of markers of oxidative stress, as well as antioxidants and oxidants, in HPV infection. Conclusion Women infected by HPV with abnormal cervical cells exhibited higher lipid peroxidation and protein oxidation, besides higher levels of GSH and GST, showing an antioxidant response to the oxidative stress induced by the viral infection and by HPV-induced cell lesions. Lastly, antioxidant modulation in cervical cells from HPV-infected women with cell abnormalities was also observed, exerted by GSH in response to lipid peroxidation. Declarations Ethics approval and consent to participate Approved by the Research Ethics Committee of the Universidade de Cruz Alta – UNICRUZ under permit no. 2.790.225, was conducted. Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analyzed during the current study are not publicly available due to individual privacy of the study participants, but are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding The present study received financial support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - under Funding code 001, and from the Programa de Apoio a Produção Científica e Tecnológica (PAPCT) of the Universidade de Cruz Alta. Authors’ contributions TMS: Conceptualization, Investigation, Resources, Methodology, Visualization, Data curation, Writing–original draft, Writing–review & editing. LK: Methodology, Investigation & Writing–review. TP: Methodology, Investigation & Writing–review. NBG: Methodology, Investigation & Writing–review. VJFF: Methodology, Investigation & Writing–review. 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Information & Authors Information Version history V1 Version 1 09 May 2025 Peer review timeline Published Cadernos Cajuína Version of Record 3 Jul 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords biochemical analysis cell cycle cellular effect human papillomavirus research and analysis methods virus classification Authors Affiliations Tatiana Mugnol Schöffel 0000-0002-1003-5800 [email protected] Universidade de Cruz Alta View all articles by this author Lara Kochenborger Universidade de Cruz Alta View all articles by this author Taiana Pedrotti Universidade de Cruz Alta View all articles by this author Nathália Billig Garces Universidade de Cruz Alta View all articles by this author Vanessa Jung Ferreira Flores Universidade de Cruz Alta View all articles by this author Janaina Coser Universidade de Cruz Alta View all articles by this author Metrics & Citations Metrics Article Usage 254 views 121 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Tatiana Mugnol Schöffel, Lara Kochenborger, Taiana Pedrotti, et al. Oxidative stress and antioxidant modulation in cervical infection by the human papillomavirus. Authorea . 09 May 2025. 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