Abstract
Background/Aim: Human papillomavirus (HPV) is the most common sexually transmitted infectious agent and, in cases of persistent infection, may cause cancer. This study evaluated the toxicological and antitumor properties of Mentha spicata extract (MSE) in KP14HPV16 mice, which carry HPV16 oncogenes.
Materials and Methods: Thirty-three female FVB/n mice (Mus musculus), including 17 HPV-transgenic and 16 wild-type (WT) mice, were divided into six groups. The control groups received tap water (WT-C, n=5, and HPV-C, n=6), while the treatment groups received either 0.50 mg/ml MSE (WT-50 and HPV-50, n=6) or 0.55 mg/ml MSE (WT-55 and HPV-55, n=5) in drinking water for 28 days. Afterwards, animals were sacrificed, and blood and organs were collected for histopathological and biochemical analysis.
Results: The main phenolic compounds in MSE were rosmarinic acid and luteolin-O-glucoronide. MSE did not significantly affect weight gain in WT mice; however, WT-55 gained significantly more weight than HPV-55. MSE demonstrated antioxidant activity as indicated by the modulation of hepatic superoxide dismutase and glutathione S-transferase (GST) activity, as well as renal GST activity, in MSE-treated HPV groups. MSE did not reduce histological lesion incidence or systemic inflammation in HPV16-transgenic mice.
Conclusion: In general, while MSE was safe and exhibited antioxidant activity, it did not significantly impact HPV16-induced lesions, warranting further research to assess systemic effects with different concentrations and durations.
Introduction
The human papillomavirus (HPV) is a nonenveloped, double-stranded DNA virus, known as the most common sexually transmitted infection globally (1). Over 170 genotypes of HPV have been found, categorized as low-risk or high-risk variants (2). High-risk variants, such as HPV16 and 18, which account for 32% of HPV infections and are responsible for 70% of cervical cancer cases (3), are implicated in cervical intraepithelial neoplasia and squamous cell carcinomas affecting the cervix, vagina, vulva, anus, penis, and oropharynx, whereas low-risk strains rarely progress to cancer but cause warts (4). Cervical cancer ranks as the fourth most prevalent cancer in women causing 341,000 deaths worldwide in 2022 (5). Despite vaccination and screening efforts, many countries lack effective protocols, highlighting the need for therapeutic alternatives (6).
Medicinal plants are valuable sources of therapeutic compounds. Spearmint (Mentha spicata L.), an aromatic plant belonging to the Lamiaceae family, grows in temperate and subtropical regions across Africa, Asia, Europe, and Oceania (7). Its leaves are widely used in both fresh and dry forms for infusions and culinary practices (8). Traditionally, it is used in the treatment of colds, coughs, asthma, fever, obesity, jaundice, and digestive problems, among many others (9). Spearmint is rich in secondary metabolites, including phenolic compounds, with antioxidant, antigenotoxic, and anticancer properties (10). Natural antioxidants, particularly dietary polyphenols, have demonstrated cancer chemopreventive potential by modulating cellular processes implicated in carcinogenesis, namely proliferation, differentiation, apoptosis, angiogenesis, and metastasis (11).
This study aimed to assess the potential chemopreventive effects and toxicological profile of a M. spicata hydroethanolic extract (MSE) in HPV16 transgenic mice, analyzing physiological parameters, histopathology of lesions, genotoxicity, and oxidative stress.
Materials and Methods
Preparation and extraction of Mentha spicata phenolic compounds. Spearmint sourced from a local producer in Bragança, Portugal (Ervital - Plantas Aromáticas e Medicinais, Lda.) was macerated in 80:20 (v/v) aqueous ethanol solution (1 g of dried plant material in 30 ml), filtered (Whatman No. 4 filter paper), and re-extracted. Ethanol was evaporated at 40°C in a Büchi R-210 rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland) and the aqueous fraction was dehydrated with a FreeZone 4.5 lyophilizer (Labconco, Kansas, MO, USA). Ten mg of MSE was dissolved in 1 ml of ethanol-water solution (20:80 v/v), and then filtered through a 0.22 μm disposable filter. The phenolic profile was determined using a Dionex Ultimate 3000, a high-performance liquid chromatographic system with a diode array detector and an electrospray ionization mass spectrometry detector (HPLC-DAD-ESI/MS) (Dionex Ultimate 3000 UPLC and Linear Ion Trap LQT XL, Thermo Scientific, San Jose, CA, USA). The capillary and source voltage were 10 V and 3.5 kV, respectively, and the capillary temperature was 175°C. Spectra were recorded in negative ion mode. The MS was programmed to carry out a series of three consecutive scans: a full mass from 150 to 1,500 Da, a zoom scan of the most abundant ion in a ±5 Da range, and an MS-MS scan of the most abundant ion in the full mass using a normalized energy of collision of 45%. The phenolic compounds were characterized according to their UV and mass spectra, retention times, comparison with authentic standards (when available), and data from the literature. For quantitative analysis, a calibration curve was obtained by injection of known concentrations of different standard compounds. The Xcalibur® data system was used to collect and process the data (Thermo Scientific, San Jose, CA, USA). Results were expressed in μg per ml of extract. MSE stability was evaluated over five consecutive days under light exposure and room temperature. Starting from day 4, the concentration of the main phenolic compounds (rosmarinic acid and luteolin-O-glucoronide) began to decrease drastically. To prevent degradation and avoid variations in the effects under evaluation, solution was replaced every 2-3 days to maintain the initial concentration.
Experimental conditions. Animal maintenance adhered to national legislation (Decree-law 113/2013, 7 August) and the European Directive 2010/63/EU, approved by the University of Trás-os-Montes and Alto Douro’s Ethics Committee (approval no. 852-e-CITAB-2020_A_1-e-122CITAB-2021) and the Portuguese Veterinary Directorate (approval no. 014139). Thirty-three female FVB/n mice (Mus musculus), aged 20-22 weeks, were used. Transgenic K14HPV16 mice, provided by Drs. Jeffrey Arbeit and Douglas Hanahan (University of California, Los Angeles, CA, USA) through the National Cancer Institute’s Mouse Repository, express HPV16 early genes in keratinized epithelia, resulting in proliferative lesions at skin and mucosal sites (4), which mirror HPV-related features in human diseases. Mice were housed in polycarbonate cages (1284L Eurostandard Type II L, Tecniplast®, Buguggiate, Italy) under controlled temperature, relative humidity, and a 12-hour light/dark cycle, with Ultragene® corncob bedding and paper enrichment. Animals were fed ad libitum with a standard diet (Mucedola® Diet Standard 4RF21, Mucedola, Settimo Milanese, Italy). Food and cages were replaced weekly or as needed. After acclimation (one week), the animals were randomly assigned to six groups: control groups drinking only water, wild type (WT-C, n=5) and K14HPV16 (HPV-C, n=6); groups supplemented with MSE at 0.50 mg/ml, wild type (WT-50, n=6) and K14HPV16 (HPV-50, n=6); and groups supplemented with MSE at 0.55 mg/ml, wild type (WT-55, n=5) and K14HPV16 (HPV-55, n=5). MSE solutions were prepared at 0.50 mg/ml and 0.55 mg/ml, representing twice the GI50 and a 10% increase over this value, respectively, based on HeLa cell proliferation inhibition (12). Body weights, as well as feed and water consumption, were recorded weekly with an analytical balance (Mettler-Toledo, Columbus, OH, USA). Ponderal weight gain was calculated using the formula (13):
Animal welfare was assessed daily using a humane endpoint table, evaluating body weight, hair condition, eye, ear, and whisker appearance, mental status, and papillomas; animals with a score equal or superior to 4 were euthanized (14). After 28 days, animals were euthanized by overdose of xylazine (20 mg/ml, Rompun® 2%, Bayer Healthcare SA, Kiel, Germany) and ketamine (100 mg/ml, Clorketam 1000, Vétoquinol, Barcarena, Portugal) via intraperitoneal injection and cardiac puncture. Blood and organs were collected for analysis. Liver, lungs, spleen, heart, and kidneys were weighed using an analytical balance (Mettler-Toledo® PM4000, Columbus, OH, USA). Relative organ weight was calculated using the formula (13):
Histopathological analysis. The liver, spleen, and ear skin tissues, fixed in 10% buffered formalin, underwent routine processing for light microscopy. Three-μm thick paraffin sections were stained with hematoxylin and eosin (H&E) and analyzed histologically. Skin epidermis was classified as normal, moderate to marked hyperplasia (increased cells and layers), dysplasia (morphological changes with moderate cytonuclear atypia), carcinoma in situ, or invasive carcinoma (15). Liver samples were assessed for necrosis and inflammation (absent, <5 multifocal inflammatory cell aggregates, or ≥5 multifocal inflammatory cell aggregates).
Hepatic and renal oxidative stress markers. Oxidative stress markers, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione S-transferase (GST), oxidized glutathione (GSSG), and lipid peroxidation (LPO), were assessed in the liver and kidney. After thawing, the samples were homogenized in cold buffer (sucrose 0.32 mM, HEPES 20 mM, MgCl2 1 mM, phenylmethyl sulfonylfluoride 0.5 mM, pH 7.4) using a TissueLyzer II apparatus (Qiagen) and centrifuged (15,000×g) for 20 min at 4°C. Supernatants were analyzed in duplicate with a PowerWave XS2 microplate scanner (Bio-Tek) or Varian Cary Eclipse Spectrofluorometer (Varian). SOD activity was measured at 560 nm, with results expressed as U/min.mg protein based on a standard curve (0-5 U/ml) (16). CAT activity was determined at 240 nm and the results were presented as U/mg protein based on a bovine catalase standard curve (0-3 U/ml) (17). GST activity was assessed based on the conjugation of reduced GSH to 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm, considering an extinction coefficient of 9.6/mM.cm (18). Results were expressed as μmol CNDB conjugated/min.mg protein. Levels of GSH and GSSG were determined through derivatization with ortho-phthalaldehyde at 320 nm and 420 nm, for excitation and emission wavelengths, respectively (19). Results were estimated using a standard curve of GSH and GSSG (0-100 μM) and expressed in μmol GSH or GSSG/mg of protein. The oxidative stress index (OSI) is the ratio between GSH and GSSG. Malondialdehyde (MDA), a marker of LPO, was measured using the thiobarbituric acid-based method at 530 nm and results were expressed in μmol MDA/mg protein (20). Protein quantification (280 nm) for normalization was performed using the Take3 Multi-Volume plate.
Genotoxicity assays. Micronucleus assay. A blood smear was made on glass slides, fixed in methanol for 10 min, and stained with Leishman’s dye (200 μl) for 2 min. Then, 400 μl of distilled water and the staining solution were added. After washing with distilled water, each slide was air-dried for 30 min and observed under a light microscope (40× objective) to count 2000 erythrocytes.
Alkaline comet assay. The procedure followed the methodology of Collins et al. (21). Thawed liver tissue was placed in phosphate-buffered saline (PBS) and cut to obtain a cell suspension, which was centrifuged (5 min, 4°C, 200×g). The supernatant was discarded, and the pellet was resuspended in 1 ml of PBS. After a second centrifugation (5 min, 4°C, 200×g), the pellet was mixed with 0.8% low melting point agarose. Three mini-gels (6 μl each) from each sample were placed on a pre-coated slide, solidified, and then incubated in a lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100, pH 10) at 4°C for 1 h. The slides were transferred to an electrophoresis tank with electrophoresis solution (10 M NaOH, 1 M EDTA) at 4°C for 30 min. Electrophoresis was performed at 24 V, 300 mA, for 25 min. Slides were washed with PBS and distilled water at 4°C for 10 min, incubated in 96% ethanol for 10 min, and dried at room temperature. Cells were stained with DAPI (1 μg/ml distilled H2O) and visualized in a BX41 Olympus microscope (400× magnification). Tail size was scored on a scale from 0 (no tail) to 4 (intense migration). For each mini-gel, 100 comets were counted. Results were expressed as genetic damage index (GDI) on an arbitrary unit (AU) scale of 0-400, calculated using the formula (13):
Statistical analysis. Data were analyzed using IBM SPSS Statistics 26 (IBM Corp., Armonk, NY, USA). Normality and homogeneity were assessed via Shapiro-Wilk and Levene’s tests. Nonparametric data were analyzed using Kruskal-Wallis with Dunn’s tests and results were expressed as medians and interquartile ranges (25th-75th percentiles), while parametric data used ANOVA with Tukey’s post hoc tests and results were presented as mean±standard deviation (SD). Statistical differences were considered when p≤0.05.
Results
The chromatographic characteristics of the compounds present in the hydroethanolic extract of Mentha spicata (retention time, λmax in UV-Vis, and mass spectral data) are presented in Table I. Thirteen compounds were identified, mostly belonging to the classes of phenolic acids (ester derived from caffeic acid) and flavonoids (flavones), which are typical of species from the Lamiaceae family and have been previously identified in this species (12). Rosmarinic acid, the most abundant phenolic compound in various Mentha species, was identified as the main compound in MSE, along with its derivatives, salvianolic and lithospermic acid (12). In addition, flavonoids like luteolin and quercetin, known for their antioxidant activities, were detected. The correlation between phenolic compounds and antioxidant activity is widely described in the literature and reinforces this extract’s bioactivity, corroborating with Sierra et al. (22) and Ćavar et al. (10).
Phenolic compounds identification and quantification (μg/ml) in M. spicata hydroethanolic extract.
During the experiment, no significant physical or behavioral changes were observed apart from the typical HPV-positive changes such as dehydration, orbital area shortening, ear papillomatosis, and chromodachryorrhea. All mice survived the 4-week experiment period, and none reached the critical limit for humane endpoint analysis (data not shown). Ponderal weight gain (Table II) was significantly higher in WT-55 compared to HPV-55 (p =0.019). No weight loss was observed in the wild-type groups with MSE supplementation. The lowest weight gain occurred in the HPV-positive groups, with the HPV-C group showing the lowest median value. Food and water consumption varied across groups; WT-55 consumed less food and drink than the other groups, while HPV-positive groups, with and without MSE, had the highest water consumption. Relative weight of internal organs is summarized in Table III. Spleen relative weight was significantly higher in HPV-C compared to WT-C (p=0.001) and in HPV-55 compared to WT-55 (p =0.010). Additionally, liver relative weight was significantly higher in HPV-C compared to WT-C (p =0.006).
Body weight variation represented as ponderal gain (%) and estimated daily drink and food consumption per animal (g).
Relative organ weights (mg organ weight/g body weight).
Hepatic and spleen lesions identified in each group are summarized in Table IV. All animals showed extramedullary hematopoiesis in the spleen. Hemorrhage was present in the spleen of HPV-positive and WT-C groups, with significant differences observed between WT-C and HPV-50 when compared with HPV-C (p=0.006 and p=0.003, respectively). Most liver lesions were observed in HPV groups, although WT-C showed all types of lesions. WT-50 exhibited different levels of hepatic inflammatory infiltrate compared to WT-C (p=0.050) and HPV-50 (p=0.046).
Number of animals (%) with histological spleen and liver lesions.
Histological results for ear skin samples are shown in Table V. Ear skin lesions were observed in HPV groups, especially HPV-C. Mild hyperplasia was found in wild type groups, particularly in WT-50 compared to HPV-50 (p =0.046), and in WT-C and HPV-C (p=0.026). Moderate to marked hyperplasia was significantly increased in all HPV groups, including WT-50 and HPV-50 (p=0.003), WT-55 and HPV-55 (p=0.002), and WT-C and HPV-C (p =0.022). Regarding dysplasia, significant differences were observed between WT-50 and HPV-50 (p=0.014), WT-55 and HPV-55 (p=0.01), and WT-C and HPV-C (p =0.022). Carcinoma in situ was found only in HPV groups, and invasive carcinoma lesions and a mast cell tumor were seen exclusively in HPV-C. Dermatitis was present in all animals, particularly WT-50 and HPV-50.
Number of animals (%) with histological cutaneous lesions.
Table VI shows the effects of MSE on hepatic and renal oxidative stress markers. In the liver, only SOD and GST activities showed significant differences. HPV-50 (p=0.003) and HPV-55 (p=0.001) exhibited lower SOD activity compared to HPV-C, while WT-50 had lower GST activity than HPV-50 (p=0.009) and WT-C (p=0.006). In the kidneys, significant differences were seen only in GST activity between HPV-50 and HPV-C (p=0.002). Micronucleus and comet assays revealed no significant changes (Table VII). HPV-55 and HPV-C had the highest average micronucleus frequency, while WT-50 and WT-C had the lowest. The highest DNA damage was seen in WT-C and the lowest in WT-55.
Evaluation of hepatic and renal oxidative stress parameters. Results are expressed as mean±SD (parametric data) or median and interquartile range (non-parametric data).
Micronucleus frequency (in 2,000 erythrocytes) and genetic damage index (expressed as arbitrary units (AU) measured in all experimental groups.
Discussion
HPV is a significant global health concern associated with neoplastic changes (23). In Portugal, infection rates rose from 2011 to 2018, predominantly among younger individuals, despite vaccination programs (24). This highlights the need for both vaccination and the exploration of new therapeutic approaches. HPV-transgenic mouse models, such as K14HPV16, are valuable tools for studying multi-step carcinogenesis (4) and assessing effectiveness of interventions (25). Mentha spicata, a species in the Mentha genus with recognized health benefits (9), has shown cytotoxic effects against various cancer cell lines, including cervical adenocarcinoma (HeLa) (12). This study evaluated the chemopreventive and toxicological effects of M. spicata leaf hydroethanolic extract (MSE) in K14HPV16 mice.
Wild-type mice supplemented with MSE exhibited greater weight gain than their HPV-positive counterparts, indicating MSE’s safety in non-HPV conditions. In HPV-transgenic mice, weight loss, primarily due to cancer cachexia, mirrors findings in literature (26). Spearmint leaf infusion, administered via intra-gastric tube, caused no significant body weight changes in Wistar albino rats (27). In this study, HPV-positive groups, irrespective of MSE supplementation, demonstrated higher drink consumption, which suggests metabolic alterations linked to HPV lesions (28). Increased relative spleen and liver weights in HPV-positive groups also reflect immune and inflammatory responses (13, 28). MSE supplementation did not mitigate organ weight changes or related histological lesions but was deemed safe under the present experimental conditions in healthy groups.
The K14HPV16 model spontaneously develops lesions due to the expression of HPV oncogenes E5, E6, and E7 (4). Histopathological analyses of ear lesions showed no significant impact from MSE, though the absence of invasive carcinoma in MSE-treated groups may indicate a potential, albeit statistically insignificant, protective effect. Flavonoids in MSE, such as rosmarinic acid, have demonstrated anticancer properties in other models, such as colorectal cancer (F344/NTac-Apcam1137 rats) (29), but these effects were not replicated in this study. Other studies have shown a preserved histoarchitecture of the testis, brain, and liver in nicotine-damaged Wistar rats after MSE treatment (30, 31).
Oxidative stress, a well-established factor in the pathogenesis of HPV-related carcinogenesis (32), can be mitigated by antioxidants found in M. spicata extracts (8, 12). For example, MSE restored nicotine-induced oxidative stress alterations (SOD, catalase, and thiobarbituric acid reactive substances) to normal levels in several tissues (30, 31). In this study, MSE was shown to modulate oxidative stress pathways, as evidenced by a significant reduction in hepatic SOD activity in MSE-treated HPV-positive groups compared to controls. Significant differences in GST activity were also observed between groups treated with MSE at the concentration of 0.50 mg/ml. In the liver, GST activity was lower in the WT-50 group compared with HPV-50, while in the kidney, GST activity was higher in HPV-50 compared with HPV-C but similar to WT-50. This aligns with previous findings in normotensive and hypertensive rats treated with rosmarinic acid (33), suggesting MSE may elicit a tissue-specific GST response. The trends observed for the remaining parameters align with the overall hypothesis of an antioxidant effect and safety of MSE.
Conclusion
This study evaluated the effects of MSE in a K14HPV16 transgenic mouse model. MSE consumption did not affect growth or cause toxicity, as no mortality, behavioral changes, or significant physiological differences were observed in treated groups. While MSE may modulate oxidative stress in HPV-related conditions, its role in mitigating HPV-induced pathogenesis remains unclear. Further studies should explore higher doses, alternative administration routes, and longer durations to better assess MSE’s therapeutic potential.
Footnotes
Authors’ Contributions
R.M.G.C. and P.A.O. conceptualized the study. T.M.J., T.A., R.S.-R., T.F., C.M., E.N.-G., J.F., A.G., M.J.P., G.L., I.G., L.F., C.V., T.C.F., and L.B. contributed to the methodology. I.G., R.M.G.C., and P.A.O. supervised the research. T.M.J., T.A., R.S.-R., and T.F. wrote the original draft. T.A., A.G., I.G., L.F., C.V., R.M., M.M.S.M.B., T.C.F., and L.B. reviewed and edited the manuscript. All Authors read and approved the final version of the manuscript.
Funding
This work was supported by national funds through FCT - Portuguese Foundation for Science and Technology: CITAB (DOI: 10.54499/UIDB/04033/2020), Inov4Agro (DOI: 10.54499/LA/P/0126/2020); CECAV, DOI: 10.54499/UIDB/00772/2020, AL4AnimalS (DOI: 10.54 499/LA/P/0059/2020), CIMO (DOI: 10.54499/UIDB/00690/2020 and DOI: 10.54499/UIDP/00690/2020), SusTEC (DOI: 10.54499/LA/P/0007/2020), LEPABE (DOI: 10.54499/UIDB/00511/2020 and DOI: 10.54499/UIDP/00511/2020), ALiCE (DOI: 10.54499/LA/P/0045/2020). T.A., R.S.-R. and T.F. thank FCT for the PhD grants 2023.01329.BD, 2022.14518.BD and 2020.04789.BD, respectively. National funding by FCT, P.I., through the scientific employment program-contract for L.F. (CEEC-IND, DOI: 10.54499/2021.00458.CEECIND/CP1690/CT0001) and L.B. (CEEC-INST, DOI: 10.54499/CEECINST/00107/2021/CP2793/CT0002).
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received January 9, 2025.
- Revision received January 21, 2025.
- Accepted January 22, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
