Potential induction of h yperkeratosis in r ats’ c ervi by gentamicin via induction of oxidative s tress,i nflammation and a poptosis

Abstract

The present study aimed to identify the possible effect of gentamicin (GEN) in Rats’ Cervi. Estradiol Valerate (EV) was used to induce cervical hyperkeratosis. GEN was administered in absence of EV. Serum and cervical GEN concentration were determined. Levels of malondialdehyde (MDA), total nitrites/nitrate (NOx), reduced glutathione (GSH), tumor necrosis factor-α (TNF-α), sirtuin type 1 (Sirt1) and nuclear factor (erythroid-derived 2)-like-2 factors (Nrf2) were measured in cervix tissue. Expression of BAX and Bcl2 were determined. Cervical histopathological examination was done. EV and GEN significantly increased MDA, NOx, TNF-α and BAX/Bcl2 ratio with decrease in GSH, Nrf2 and Sirt1 levels in cervical tissue. Histopathological picture of diffuse and marked hyperkeratosis was detected in EV and GEN groups. In conclusion, GEN-induced cervical hyperkeratosis via induction of oxidative stress, inflammation and apoptosis.

Keywords

Hyperkeratosis gentamicin estradiol sirtuin type 1 nuclear factor (erythroid-derived 2)-like-2 factors

Introduction

Hyperkeratosis is a benign structural alteration of the cervical squamous epithelium that might conceal dysplastic lesions and make accurate colpo-cytological evaluation difficult.¹ A colposcopy allows the cervix and the tissue on its surface to be examined more closely for example, cervical cytology collects cervical cells so they can be checked for changes caused by HPV which if left untreated may be turn into cervical cancer.² Hyperkeratosis describes a phenomenon,³ in which cervical squamous epithelial cells turn to jacinth after dyeing. The increased thickness of the stratum corneum, the skin's outer layer, is referred to as hyperkeratosis. It is most usually caused by chronic physical or chemical injury, such as friction or the use of aggressive soaps. Keratosis can be brought on by a number of external or endogenous causes and is associated with increased keratin production. The majority of the time in clinical practice, it is caused by long-term physical or chemical trauma such friction or the use of harsh soaps (especially those with a basic pH), but it can also be caused by long-term inflammation or a side effect of many medications, including chemotherapy.⁴ So, cervical hyperkeratosis is a common gynecological lesion and usually caused by inflammation or trauma.³

Gentamicin (GEN) an aminoglycoside antibiotic, is bactericidal. It passes through the gram-negative membrane in an oxygen-dependent active transport as it binds to bacterial ribosomes and inhibits protein synthesis.⁵ GEN is a commonly used drug in many systemic, gynecological and obstetric infections. In a previous study, the agar diffusion method was used to determine the levels of GEN in the blood, menstrual fluid, cervical mucus, and vaginal fluid of women who were receiving it. A significant level of GEN was found in menstrual fluid, cervical mucus and vaginal fluid that was comparable to its serum drug levels.⁵ Furthermore, certain infections are more common in females with a higher susceptibility to GEN such as acute pyelonephritis. In these infections, as fluoroquinolones, cephalosporins, and many other classes of antibacterials face an era of rising antimicrobial resistance, GEN is being investigated as an alternate antibiotic choice for early empirical therapy, especially in regions with limited medical resources or where the rate of antibiotic resistance among uropathogens is high.⁶

Nuclear factor (erythroid-derived 2)-like-2 factors (Nrf2) has a critical role in elimination of free radicals through its cytoprotective effects against oxidative stress and inflammation.⁷ Nrf2 is considered a marker of poor prognosis in cervical cancer, it protects normal cells from transformation but also promotes proliferation and survival.⁸ Nrf2 signaling pathway has an important key role in anti-oxidation and anti-inflammation in many chronic diseases and complications.⁹ On the other hand, Sirtuin type 1 (Sirt1) -a member of the vast Sirtuin family-which has a histone deacetylase activity resulted in regulation of cell death/survival and stress responses.¹⁰ There are significant correlations between SIRT1 expression and risk factors for endometrial carcinoma. SIRT1 is considered a target for the effective treatment of cervical cancer.¹¹ Modulation of these parameters could explain how GEN can induce cervical hyperkeratosis. SIRT1/Nrf2/TNF-α signaling mediates oxidative stress and inflammation.¹²

Aim of the current work

Consequently, in the current study, we aimed to extend the work and investigate the effect of GEN in Rats’ Cervi as GEN is a commonly used antibacterial agent with a variety of uses. Cervical hyperkeratosis as an important cause for false negative results of Pap smear and human papillomavirus test

Materials and methods

Ethics

Rats were processed and rehabilitated in accordance with the Institutional Ethical Committee's (Faculty of Medicine, Minia University, Egypt) guidelines for experimental animal care, as well as the National Institutes of Health's (NIH) Guide for the Care and Use of Laboratory Animals (Approval number:393:2022).

Drugs

GEN was purchased from Memphis pharm & chemical Ind. Egypt. Estradiol Valerate (EV) was obtained from Bayer pharma AG, Germany.

Animals and experimental design

Twenty-four female Wistar rats weighing 180-210 g and aged 8-10 weeks were obtained from Egypt's National Research Center in Cairo. Before beginning the experiment, rats were acclimatized in their cages (4 rats/cage) for 1 week in a normal light-dark cycle with free access to tap water and a normal meal (El-Nile Company, Egypt). The study lasted 1 week, and the animals were divided into three groups of eight rats each, as shown below: Control group; rats received carboxymethyl cellulose (CMC) for 1 week, EV group; rats administered EV (2 mg/kg/day, p. o.) for 1 week,¹³ GEN group; rats administered GEN (100 mg/kg/day, i. p.) for 1 weeks.^14,15

Sample collection

Rats were scarified at the end of the experiment, and their cervi were removed and cleaned with saline to remove any blood. A section was preserved for histological research. The other part was homogenized with phosphate buffer (0.01 M, pH 7.4; 20% w/v) (tissue weight (g): phosphate buffer (mL) volume = 1:5). To measure the biochemical parameters, the homogenate was centrifuged for 15 min at 5000 r/min, and the supernatant was kept at −80° C.

Biochemical investigations

Measurement of serum and cervical GEN level

Serum and cervical GEN concentration were measured with the commercial rat ELISA kit; Cell biolabs, California, USA (Catalog No: MET- 5135).

Determination of oxidative stress parameters

Malondialdehyde (MDA) is a lipid peroxidation indicator. The sample absorbance was evaluated at 535 nm using the thiobarbituric acid technique.¹⁶ Total nitrite/nitrate (NOx) was determined using the Griess reaction between nitrite and a mixture of naphthyl ethylenediamine and sulfanilamide; the NOx level was detected at 540 nm.¹⁷ Reduced glutathione (GSH) was determined using commercial kits (Biodiagnostic, Egypt) as guided by the manufacturer.

Assessment of Sirt1, Nrf2 and TNF-α levels

TNF-α (Elabscience Biotechnology Inc., USA, E-EL-R2856), Sirt1 and Nrf2 levels (MyBioSource, Inc., San Diego, USA Catalog No: MBS2601957, MBS775081, respectively) were determined using ELISA kit.

Real-Time reverse-transcription polymerase chain reaction of BAX and Bcl2 gene expression

Ribozol reagent (Ameresco, Solon, USA) was used to extract total RNA. The total quantity of RNA was detected at A260 nm, and the RNA purity was determined using the ratio of A260 to A280. RNA samples with purity more than 1.7 were utilized for RT-PCR. Equal amounts of total RNA were reverse transcribed into cDNA using a RevertAidTM First Strand cDNA Synthesis kit (Fermentas, Life Sciences). In the Real-Time PCR Detection System, RT-PCR was performed using SYBR Green QPCR Mix (Solis, BioDyne). The primers sequences, were obtained from NCBI, are as follows: BAX forward, 5′-GGAGACACCTGAGCTGACCT-3′ and reverse, 5′-CTCAGCCCATCTTCTTCCAG-3′. Bcl2 forward, 5′-TATATGGCCCCAGC ATGCGA-3′ and reverse, 5′-GGGCAGGTTTGTCG ACCTCA-3′. GAPDH forward primers are as follows: 5′-GTCGGTGTGAACGGATTTG-3′ and reverse 5′-CTTGCCGTGGGTAGAGTCAT-3′. The formula 2 ^(−ΔΔCt) was used to evaluate the relative gene expression level of each gene after being normalized to that of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference gene. The outcomes of each experimental sample were thus represented graphically as relative expressions in comparison to the control.

Histopathological study

The female rats’ cervi were dissected and immersed in a 10% formalin solution for 24h, then processed and embedded in paraffin. Cross-sections 5μm thick were cut using a microtome. These tissue sections were stained with hematoxylin-eosin stain and observed under an Olympus light microscope to be histopathologically evaluated. Hyperkeratosis was classified into four scores: score 0 represents absent hyperkeratosis; score 1 represents mild hyperkeratosis; score 2 represents moderate hyperkeratosis and score 3 represents marked hyperkeratosis.

Statistical analysis

All values were expressed as mean ± SEM. The statistical analyses were conducted using GraphPad Prism (version 5.0; San Diego, CA, USA). Analysis of variance (ANOVA) for multiple comparisons, followed by the Tukey–Kramer test as a post-ANOVA test, was used for data analysis. The results were considered statistically significant if the p-value was <0.05.

Results

Impact on serum and cervical GEN concentration

There was significant increase in both serum and cervical GEN concentration in the GEN group when compared to control and EV groups as shown in Figure 1(a) and (b) respectively.

Figure 1.

Impact on serum and cervical GEN concentration. Data represent the mean ± SEM. p-value <.05 is set for significance. (a) Significant difference from the control group. (b) Significant difference from the EV group [EV: Estardiol Valerate; GEN: gentamicin].

Impact on cervical oxidative stress parameters

As shown in Table 1, EV and GEN significantly increased MDA by 3.25%; 3.44% (p value <.0001) (37.91 ± 2.74; 40.05 ± 2.92), respectively and NOx by 1.82%; 1.78% (p value <.0001) (103.1 ± 3.43; 100.6 ± 3.69), respectively and reduced GSH by 0.41%; 0.42% (p value <.0001) (27.16 ± 2.54; 27.38 ± 1.43), respectively as compared to control group (MDA: 11.63 ± 0.70; NOx: 56.40 ± 2.81; GSH: 65.93 ± 4.68).

Table 1.

Impact on oxidative stress parameters.

Groups	Cervical MDA (nmol/g tissue)	Cervical GSH (nmol/g tissue)	Cervical NOx (µmol/g tissue)
CONT	11.63 ± 0.70	65.93 ± 4.68	56.40 ± 2.81
EV	37.91 ± 2.74^a	27.16 ± 2.54^a	103.1 ± 3.43^a
GEN	40.05 ± 2.92^b	27.38 ± 1.43^b	100.6 ± 3.69^b

Results represent the mean ± SEM (n = 8).

^aSignificance from the control group;

^bSignificance from EV group. [EV: Estradiol Valerate; GEN: gentamicin; MDA: malondialdehyde; GSH: reduced glutathione; NOx: total nitrite/nitrate].

Significance is at p < .05.

Impact on cervical TNF-α, Sirt1 and Nrf2 levels

In groups received EV and GEN, TNF-α level (p value <.0001) (46.58 ± 2.84; 48.32 ± 3.04) was increased significantly by 3.5%; 3.7%, respectively with significantly reduction of Sirt1 level by 0.37%; 0.39% (p value <.0001) (24.29 ± 1.75; 25.34 ± 2.34), respectively and Nrf2 level by 0.48%; 0.50% (p value <.0001) (48.73 ± 2.58; 51.40 ± 3.83), respectively as compared to control group (TNF-α: 13.10 ± 1.12; Sirt1: 64.95 ± 3.78; Nrf2: 101.0 ± 3.79) (Figure 2).

Figure 2.

Impact on Sirt1, Nrf2 and TNF-α. Results represent the mean ± SEM (n = 8). (a) Significance from the control group; (b) significance from EV group. [EV: Estardiol Valerate; GEN: gentamicin; Sirt1:Sirtuin type 1; Nrf2: nuclear factor (erythroid-derived 2)-like-2 factors; TNF-α = tumor necrosis factor alpha]. Significance is at p < .05.

Impact on cervical BAX, BCL2 and BAX/BCL2 ratio

As presented in Figure 3, EV and GEN significantly increased BAX by 2.9%; 2.94% (p value <.0001) (2.90 ± 0.19; 2.94 ± 0.19), respectively and reduced BCL2 by 0.66%; 0.59% (p value <.0001) (0.66 ± 0.046; 0.59 ± 0.05), respectively as compared to control group. Subsequently, EV and GEN significantly increased BAX/BCL2 ratio by 4.6%; 5.16% (p value <.0001) (4.61 ± 0.52; 5.16 ± 0.44), respectively when compared to control group (1.00 ± 0.00).

Figure 3.

Impact on mRNA expression of BAX (a), Bcl2 (b) and BAX/Bcl2 ratio (c). Results represent the mean ± SEM (n = 8). (a) Significance from the control group; (b) significance from EV group. [EV: Estardiol Valerate; GEN: gentamicin]. Significance is at p < .05.

Histopathological results

Cervi of groups received EV or GEN showed marked and diffuse areas of stratified squamous keratinized epithelium (hyperkeratosis). Meanwhile, cervi of control group lined by stratified squamous non keratinized epithelium (Figure 4). Table 2 showed histopathological scoring.

Figure 4.

Effect on histopathological picture of rats’ cervi. (a) Control group cervi are lined by stratified squamous non keratinized epithelium. (b, c): EV and GEN groups, respectively, cervi show diffuse and marked hyperkeratosis (x 200). EV; Estardiol Valerate, GEN; gentamicin, (n = 8).

Table 2.

Histopathological scoring.

Groups	Histopathological scoring
CONT	0.25 ± 0.16
EV	2.63 ± 0.18^a
GEN	2.75 ± 0.16^b

Results represent the mean ± SEM (n = 8).

^aSignificance from the control group;

^bSignificance from EV group. [EV: Estradiol Valerate; GEN: gentamicin].

Significance is at p < .05.

Discussion

In both clinical and practical terms, hyperkeratosis refers to the formation of extra keratinaceous material on the skin's surface, which may appear as scale or another type of keratinaceous buildup. Histology defines hyperkeratosis as an abnormally thick stratum corneum that typically exhibits aberrant keratinization as parakeratosis, compacted orthokeratosis, or a combination of the two.¹⁸

A microscopic layer of thickened parakeratin and/or orthokeratin of the mucosal epithelium in hyperkeratosis situations appear. Because the increased keratin layer has a whitish clinical appearance in the moist environment of the mucus membranes, the word hyperkeratosis is frequently used clinically to refer to white regions on mucosa without a diagnosis. Chronic irritation is the most common cause of hyperkeratosis. These lesions must be watched since they may be cancerous in certain circumstances.¹⁹

In both women and laboratory animals, mature nonsquamous epithelium is replaced by stratified squamous epithelium of the endocervical canal. Endocervical squamous metaplasia has been found in rats and primates following estrogenic drug administration. Irritation features such as epithelial exfoliation, hemorrhages, edema, mucosal necrosis and inflammation, ulceration, atrophy, and hyperplasia are assessed at both the macroscopic and microscopic levels.²⁰

In the current study, estradiol was used as a positive control drug that induced cervical hyperkeratosis as mentioned in previous studies and confirmed by typical histopathological changes.^3,20

To the best of knowledge, this is the first time to report occurrence of hyperkeratosis with GEN administration. However, it was reported that GEN exists in cervical mucus in a considerable concentration that is comparable to serum level.⁵ As mentioned before, hyperkeratosis may be related to chronic irritation. In the case of GEN, induction of oxidative stress, inflammation and apoptosis occurred as shown by increase in MDA, NOx, TNF-α, BAX, and BAX/Bcl2 ratio with reduction in GSH, Sirt1, Nrf2 and Bcl2. These actions were parallel to changes in histopathological picture that denoted marked and diffuse areas of hyperkeratosis in cervical tissue. Current findings are supported by reports confirmed that GEN caused induction of oxidative stress, inflammation and apoptosis in many organs such as kidney, ear, liver and testis.^21–25

In these studies, Oxidative stress seems to be a principal molecular mechanism in tissue injury induced by GEN. GEN directly enhances the generation of mitochondrial reactive oxygen species (ROS) which cause deleterious effects on biomolecules such as proteins, lipids, and nucleic acids. Furthermore, ROS overproduction promotes apoptosis and inflammation.²⁶ Excessive oxidative stress depletes Nrf2, the cytoprotective antioxidant protein.⁶ Oxidative stress upregulates synthesis of proinflammatory cytokine such as TNF-α and IL-1β that suggest a significant mechanistic link between oxidation and inflammation.²⁷ TNF-α is negatively influenced by Nrf2, indicating that GEN aimed to activate inflammation and hyperkeratosis. In addition, Sirt1, a NAD⁺-dependent protein deacetylase, regulates such cellular processes as stress response and longevity.²⁸ In the present study, GEN inhibits Sirt1 activity. Sirt1 activated the Nrf2 pathway affecting ROS production through advanced glycation end products.²⁹ N-terminal domain of Sirt1 is involved in the deacetylation of caspase-3. It suppresses caspase-3 activation directly. So, Sirt1 affect apoptosis also.³⁰ All of these demonstrate that Sirt1 is a corner stone in antioxidant, anti-inflammatory and anti-apoptotic defense systems regulation. Contributing to cervical hyperkeratosis induced by GEN, further studies are strongly recommended to examine GEN in different doses and its ability to cause cervical hyperkeratosis. The induction of hyperkeratosis by GEN is supported by previous studies stated that tissue dysfunction induced by GEN was accompanied by enhancement of oxidative stress and inflammation statuses in various organs like kidney, liver, ear, testis and nervous system.^24,31–34

Conclusion

GEN is a commonly used antibacterial agent with a variety of uses. Clinical studies in human are necessary to validate the effect of GEN in inducing cervical hyperkeratosis. Molecular mechanisms underlying some of pharmacological effect of GEN, still remain unclear and need to be further investigated.

Footnotes

Authors' contributions

Walaa Yehia Abdelzaher, Remon Roshdy Rofaeil, Mohamed Ahmed Zeen El-Din, Alaa gamal ElSetae participated in conceptualization, performing the experiments, data analysis, editing, and revising the manuscript. Mina Ezzat Attya performed and wrote the pathological examination. Hatem A. Mohamed performed and wrote PCR. All the authors read, revised, and approved the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Walaa Y ehia Abdelzaher

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