The possible ameliorative effect of Resveratrol on the lead exposed parotid glands in albino rats: Insights into glandular histology and DNA damage

Introduction

Lead (Pb) is a part of chemical components that produce significant threat to wellness and it is yet still remain a health concern due to its prevalent usage (1). Pb is diversified distributed in the human body and his effect is proportional to its dose and time (2). Some organs in the body, have higher susceptibility for lead to be deposited inside like the kidney; neural; immune; genital and circulatory system (3); in addition, bone marrow, bones and dental tissues are also more vulnerable to lead toxicity (1).

Various workers in the Lead manufacturing are ignorant of the detrimental impacts of lead and doesn’t avoid the risk of lead handling, giving rise to heightened predisposition to toxicity. Lead fabrication involve; lead manufacturing and bonding, making of porcelain, batteries, plastics and shot, additionally for restoring the metal heater and reusing the lead wires. Workers in some jobs like destruction of old buildings, scratching the lead paints, restoration of bridges, storage tanks, connection between the metallic coated lead and furniture refinishing might also be exposed to significant load of lead (4).

Over the past few years, one of the main mechanisms explained the lead toxicity is a derangement in the oxidant-antioxidant ratio, thus provoking oxidative insult in cells with the propagation of large number of free radicals (ROS), for instance hydroxyl radical, hydrogen peroxide, hyperoxide and lipid oxidation products (5). It is noteworthy that salivary glands of experimental animals are vulnerable to damage when subjected to metals. Many latest findings have revealed that besides biochemical and proteomic changes, exposure to metals such as aluminium and mercury induced damages on acinar and myoepithelial cell cytoskeleton integrity, volumetric changes, and cell death. Moreover, metallothionein (protein contribute to the reclamation of heavy metals) was found to be a biosensor of the destruction occurred as a result of the inflow of metal (6). Any deterioration of salivary gland function will influence both the oral health and other systems in the body mainly digestive tract because they are accountable for the formation of saliva and necessary for the conservation of internal equilibrium of the oral cavity's environment (7).

To date the molecular pathways connecting lead exposure and dental disease are not fully understood, it was suggested that lead disturb salivation, consequently heightened the risk of dental caries. Experimental researches reported that lead exposure during the gestational period can produce remarkably higher levels of dental caries with noticeable decrease in salivary secretion (8). Comparably, postnatal ingestion of lead in experimental animals conveyed reduction in salivary Ca+2 and protein amount, higher oxidative degradation of lipids with decline in the whole antioxidant power suggesting that production of oxidative stress (9).

Resveratrol (RV) (trans-3,40,5-trihydroxystilbene), a naturally occurring polyphenolic, nonflavonoid free radical scavenger and a phytoalexin compound found in many food items, especially in grape skins (50–100 lg/g) and red wine (1.52 mg/L) (10). Resveratrol has been proven to have outstanding immune-regulating, antioxidant, and anti-inflammatory effects (11). The antioxidant properties of resveratrol are largely depending on the enhancement of redox homeostasis system with direct ROS-scavenging effects (12).

Moreover, resveratrol able to bind with many cells Ligands, for instance estrogen synthase, P-glycoprotein (P-gp), DNA polymerase, bacteriophage T4 gyrase, oestrogen receptors, tubulin, and ATP synthase. As well it has the ability to trigger a lot of sequence-specific DNA-binding factors, inhibit the appearance of antiapoptotic gene products, reduces protein kinases, persuade endogenous antioxidant defense system, decrease the release of inflammatory mediators, impede the appearance of angiogenic factors and metastasis-associated proteins and genes, and adjust tumor suppressor genes. Consequently, this polyphenol has promising effects to defeat cancer (13).

The single-cell gel electrophoresis (SCGE), the comet assay, is a novel approach for assessment of DNA damage (14). Its core concepts depend on the movement of DNA out of the nucleus by using electrophoresis in an agarose gel. Then when view the cells under microscope it will appear like a comet with a head representing the nucleus area and a tail which containing DNA segments or strand displacement (15). It is fast and easy way to measure the DNA damage, besides it only needs small number of cells in each sample and provide data about the individual cell level so it is widely used and preferred over the other methods (16).

On the basis of this condition, by investigating the histologic findings, apoptotic and DNA changes of parotid glands in an experimental design, we focused to explore the probable preservative consequences of resveratrol in opposition to the degeneration of rats’ parotid gland administered lead.

Materials and Methods

Ethics approval and consent to participate

All research steps were approved by the Mansoura University animal care and use committee, Egypt, approval No. MU-ACUC (DENT.R.25.04.23). This assessment was carried out in adherence to the ARRIVE (Animal Research: Reporting of in Vivo Experiments) guidelines and regulations (https:// arriveguidelines.org). The maintenance and care of the experimental animals followed the International Guiding Principles for Biomedical Research Involving Animals.

Animals

Forty-five adult male albino rats, aged 4 months and weighing 200–250 grams, were utilized in the research. The rats were confined in separate cages, provided with a balanced diet, and maintained under secure and appropriate conditions.

Following 1 week of adaptation, the rats were haphazardly divided into three groups each contain 15 rats:

1. Group I (control group): 15 rats were received 50 ml/kg/day of deionized water by gastric intubation for 55 days.

1. Group II (Lead Group): 15 rats were received 50 mg/kg/day of lead (Pb) acetate (Sigma-Aldrich, Germany) by gastric intubation for 55 days (17).

1. Group III: 15 rats were received 50 mg/kg/day of lead (Pb) acetate by gastric intubation for 55 days and simultaneously, 10 mg/kg/day of resveratrol (RSV) (resVida, DSM, Basel, Switzerland) intraperitoneally for 28 days (18).

Sample size: 45

N value: 15

The sample size and N value was planned based on exact considerations including the significance level (P value), sensitivity (1 − β) of the research, minimal clinically relevant difference and the incidence rate

Histological evaluation

Following the treatment with lead acetate in group II and treatment with resveratrol in group III, the rats were terminated by an overdose of fluothane. Parotid glands were promptly removed and prepared for:

1. H&E and immunohistochemical staining by placing in 4% buffered paraformaldehyde (pH 7.4) for half a day. The water in the specimens were removed out by ascending grades of ethanol then immersed in molten paraffin wax. The wax blocks were sectioned into 5 to 6 µm slices using a fully automatic microtome (RMC Products, Tucson, AZ, USA). For light microscopic examination by using light microscope (Olympus CH2; Olympus Optical Co., Ltd., Tokyo, Japan).

1. sections of each sample were stained with hematoxylin and eosin (H&E), and for immunohistochemical examination by caspase 3. Immunohistochemical changes observed through the microscopy were recorded based on the results as follow: negative or none, mild, moderate, and intense (19).

1. The comet assay, the excised glands were maintained in 1ml saline solution in microcentrifuge tube and persist in being iced at -180c (20,21).

Immunohistochemistry evaluation

To perform the immunostaining the wax firstly removed from the sections, hydrated, and then incubated in 3%H2O2 for 20 min for peroxidase blockage. At that time, the sections were embedded in goat serum for 2 h, and then incubated in rabbit anti-rat Cu-Zn SOD multiclonal antibody (1:500 dilution; Santa Cruz) and rabbit anti-rat Casp-3 multiclonal antibody (1:100 dilution; Beijing Zhongshan Golden bridge Biotechnology Co, Ltd) at 4°C for 24 h. Then the biotin-conjugated goat anti-rabbit IgG and ABC solution (Beijing Zhongshan Golden bridge Biotechnology Co, Ltd) were overlaying the sections sequentially, and each reagent was allowed to react at standard temperature for 3 h. lastly, the sections were stained with DAB kit, and the immunoreactive products were stained brown. The sections of control group were managed as described above in the same manner, but, the rabbit anti-rat Cu-Zn SOD and Casp-3 polyclonal antibody were exchanged by 0.1 mol/L PBS (22).

Comet assay methodology (20,21)

The Comet assay also called single cell gel electrophoresis is a rapid and very sensitive fluorescent microscopic method to assess DNA damage and repair in individual, nucleated cells. In this test, cells are disabled in a bed of low gelling temperature agarose, succeeded by gentle cell lysis, treated with alkali to uncoil and separating the strands of DNA, and hydrolyze sites of damage. The specimens are then electrophoresed and stained with a fluorescent DNA intercalating agent. The specimens are then visualized by wide-field fluorescence microscopy. Its main concept depends on the assessment of the denatured, cleaved DNA fragments to move out of the nucleus under the influence of an electrostatic field, when a current is applied the intact DNA moves slower and remains within the nucleoid region. The DNA “comet” tail shape and migration pattern is directly proportional to the amount of DNA damage.

Statistical analysis

Statistical analysis was done to determine the differences between the three groups. Data are presented as mean ± standard deviation of the mean (SDM). The data were analyzed using Statistical Package for the Social Sciences (SPSS) version 20.0. One-way ANOVA test was done followed by post hoc test. For all statistical tests, P ≤ 0.05 was taken as the level of significance.

DNA fragmentation pattern was estimated, in which the single cell gel electrophoretic slid is alongside with a computer-assisted image analysis system (Comet Assay IV software, Perspective Instruments). 100 haphazardly chosen cells / slide were recorded. The DNA deterioration was assessed by evaluating the ratio of tail DNA content in relation to the whole cellular DNA content.

Selection criteria:

The choice of the model organisms for the study depends on the following considerations: 1) practicality as an analog, 2) communicability, 3) genetic consistency, as needed, 4) knowledge about biological characteristics, and 5) price and accessibility.

Disqualifiers:

The animals were precluding if obstacles were anticipated during parotid gland excision or there was a failure to reach quality assurance, such as undesirable levels of contaminants or suboptimal histological quality.

Results

Histological results:

Examination of H&E-stained sections showed that group (I) had the classic architecture of the parotid gland appeared in the form of serous acini. The acini appeared rounded with secretory cells have large rounded vesicular basophilic nucleus. Narrow lumina and apical acidophilia was evident. Fig. 1 (A)

While, stained sections of operated group (II) revealed the loss of acinar cell boundaries with darkly stained nuclei and many vacuoles in their cytoplasm, condensation of chromatin in the nucleus and distended and clogged blood vessels. Expanded intralobular ducts were also found. Destructed cells with breakage membrane appeared in the acini and ducts. Signs of dysplasia were also noted in the form of abnormal variations in size and shape of nuclei, nuclear hyperchromatism and nuclear enlargement in comparison to cytoplasm Fig. 1 (B)

In the stained sections of group (III), to some extent, normal appearance of serous acini and ducts was seen but with little vacuoles in the cytoplasm. The ducts appeared with regular walls and narrow lumina. Fig. 1 (C)

Immunohistochemical results:

The stained specimens seen by using the optical microscope by experienced researcher. Histopathological scoring systems (negative/mild/moderate/intense) are frequently used to transform personal interpretation of immunohistochemistry (IHC)-marker appearance into approximate values, which subsequently used for data investigation and set up the conclusions. The clinical scoring system depends on two features: the general staining intensity and the ratio of stained tissue or cells.

The immunopositivity results were seen as brown deposits by using DAB (3,3'-diaminobenzidine) chromogen stain. The appearance of caspase 3 was considered to be positive if there was any staining of the cytoplasm, irrespective of staining intensity, fig. 1 (D), (E), (F) and fig. 2 - table 1.

Group I (Control group):

The serous acinar cells showed mild reaction of caspase 3 in the cytoplasm of serous acini, Fig. 1 (D).

Group II:

There was intense reaction of caspase 3 in the acinar cells, Fig. 1 (E).

Group III:

There was moderate reaction of caspase 3 in the cytoplasm of the acinar cells, Fig. 1 (F).

Comet assay analysis

Comet assay was conducted to study the degree of genotoxic damage in the acinar cells of all groups. In group I, most of cells seemed to be in the form of uniform circles with a slight tail moment arm, little % of tail length and tail DNA.

In group II, number of tailed cells and the tail length was highly increased in comparison to group I. In group III, the tail moment seemed to be remarkably declined in contrast to that found in the group II, fig. 3.

The predicted values of comet assay results were measured for parotid gland cells of all groups: tail moment, tail DNA %, tail length, untailed and tailed, fig. 4.

The standard error of the mean for comet assay variables and its statistical significance in the parotid gland for the different groups were presented in table 2.

Discussion

Lead is a naturally occurring, bluish-grey metal found in trace quantities in the crust of the planet (23). Due to its non-biodegradable nature and continued usage, its concentration increases in the environment, posing a growing risk to human health (24). It is the most dangerous heavy metal present in the environment. The United States Agency for Toxic Substances and Disease Registry (ATSDR) ranked lead as the second on the priority list of toxic substances (25). Lead is absorbed by inhalation, ingestion, and contact with the skin. Nonetheless, the bulk of lead enters the body via the digestive system through contaminated food and drink (26).

Parotid gland (PG), the largest major salivary gland, may be affected structurally and functionally by various factors leading to toxicity (27). Because many significant protein units, like the amylase enzyme and epidermal growth factor, are secreted from the salivary glands (28). It is urgent to discover the technique of lead toxicity on the salivary glands and how to avoid these adverse effects.

In the current study, ingestion of Pb by gastric tube intended to imitate to the way that human also could exposed to lead toxicity which primarily occurs by gastrointestinal absorption. Even though, in this study the amount of lead toxicity were not measured after chronic exposure, its mechanism of action stated that once distributed in the blood flow, 30 to 40% of Pb remains concentrated in blood cells, and subsequently settled in many organs such as liver, kidneys, lungs, brain, bones, and teeth for the next 4 - 6 weeks (29). In addition, lead carbonate (PbCO3) or Lead (II) carbonate (PbCO3Cl-) have the capability to diffuse through plasma membranes and collected in organelles such as mitochondria, endoplasmic reticulum, and secretory granules (30).

The findings of the current research provided verification that lead (Pb) produces diverse types of deterioration in parotid gland of group II. Light microscopic examination of the parotid gland sections of group II revealed aggravating parotid architecture with deterioration of the acinar cells. These results were in agreement with (Nourhan M. et al., 2022) who investigated the impact of lead on parotid and submandibular salivary glands and stated the confirmation of the cell toxicity of lead, involving apoptotic bodies and damaged mitochondria, on the other hand, stuck secretion were noticed in the ducts lumens (31). Moreover, they proved that rats treated with lead acetate were shown to have altered secretions of protein, calcium, and the lysosomal enzyme N acetyl-/-D-glycosaminidase (NAG) (32). In addition to decreasing the level of calcium and protein in saliva, elevating lipid oxidation, and decreased total antioxidant power in the acinar cells. This suggested the presence of lead-induced oxidative damage (33).

Lead poisoning occurred as a result of its binding affinity to the thiol group (–SH) of some proteins and enzymes that leads to progressive alterations in structure and function of cells and induce cellular dysfunction (1). Potentially attributable to the binding of Pb2+ to Ca2+ receptors on the plasma membrane maintaining its function thus enhance more flow of Pb2+ intracellularly (34); hence, metal accumulation occur and produces the negative impacts on the cell metabolic processes (35). One more principal toxicodynamic related to Pb2+ that it persuade redox imbalance i.e. a disproportion between formation of (ROS) and the antioxidant power of various biological molecules such as proteins, lipids, carbohydrates, and DNA (36).

Moreover, Lead can give rise to cellular and biological injury to the parotid gland (PG) and submandibular gland (SMG) in addition to the advancement of glandular oxidative stress. Meaning that the flow of Pb intracellularly influences the microfilaments of the myoepithelial cell’s skeleton, that are pivotal for gland contraction and salivation, this was evidenced by the diminished immunoreactivity of cytokeratin (CK‑19) in both the PG and SMG, clarifying the reduction of salivation. It is worth noting that ductal injury may also influence saliva formation (37).

Caspase-3 is a frequent activated death protease indicating cellular suicide generating the specific break up of many crucial cellular proteins (38). As a consequence, it is dominant for specific procedures concerned with the break apart of the cell and the formation of apoptotic fragments, but it may also function before or at the stage when adherence to cell death is made (39).

In the ongoing research, glands of the control group stained with anti-caspase 3 showed a mild reaction in the cytoplasm of the acini, while in group II, the reaction was intense. Results for group III showed a moderate reaction of anti-caspase 3 in the cytoplasm of the acinar cells in comparison to group II, indicating a reduction in the amount of apoptosis. The mentioned detections come in consonance with Laila E Amin et al., 2016, who studied the effect of cadmium toxicity on major salivary glands. Cadmium and other heavy metals like lead induce cell apoptosis mostly in the parotid greater than submandibular glands (40).

Constantly, one more clarification was introduced by Permentar et al. 2011, observed the impact of heavy metals on rats hepatocytes cell lines; they recommended that heavy metals disturbed the genes related to endoplasmic reticulum stress and increase the unfolded protein response resulting in programmed cell death (41).

The SCGE (comet) assay determined to be an extremely precise way for genotoxicity testing (42). Due to its accuracy to recognize genomic damage at the single cell level and its potential application to virtually all eukaryotic cells types, the assay has quickly been adopted as useful tool in short term genotoxicity and human biomonitoring studies (43).

In this research, the alkaline comet assay results of in the reference group revealed most of cells has equivalent circle shapes with a minimal tail moment, slight tail length and tail DNA % suggesting minimal DNA damage, while in group II, the percentage of DNA in the tail, tail length and tail moments was significantly higher than group I pointing to greater oxidative DNA damage as a result of lead intoxication, and these outcomes were congruent with K Danadevi et al., 2003 whoever investigated the genotoxic impact of Pb exposure in the workers exposed to lead using comet assay. DNA damage appeared in their blood leucocytes, directly correlating with higher blood lead levels and years of exposure, confirming the genotoxic effects of occupational lead exposure and highlighting the sensitivity of the comet assay for biomonitoring (44).

Furthermore, Mohammad Arif et al., 2008 reported the same findings of DNA damage in rats’ erythrocytes managed with lead as evaluated by comet assay; they proved that administration of lead is related to generation of redox imbalance giving rise to oxidative DNA lesions. As a consequence, genotoxic stress in the circulating blood could be an appropriate early warning signal to observe the environmental or employment lead poisoning (45).

Numerous investigations illustrated that resveratrol complement straightway quench the liberation of Inflammatory cytokines including tumour necrosis factor α (TNFα), interleukin β (IL-β), IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), interferon α (IFNα) and IFNβ in a wide range of tissues (46). Resveratrol also displays antioxidant activity in cell culture, which contributes to a reduction in inflammatory responses. A common link between the inhibitory effects of resveratrol could be due to its ability to inhibit factors involved in gene transcription such as mitogen-activated protein kinase (MAPK), C-Jun N-Terminal Kinases (c-JNK), Activator protein-1 (AP-1) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (47).

In the current research, group III treated with resveratrol and stained with H&E presented regaining of the plain-looking of acinar cells and ducts and this indicates the antioxidant and precautionary impact of resveratrol in opposition to the lead toxicity. Xiang Yi et al., 2025 approved the same findings when they researched the resveratrol influence on HT-22 cells; mouse hippocampal neuronal cell line which subjected to lead, they noticed that lead (200 μmol/L) lessen the BCL2 protein synthesis, at the same time rising poly (ADP-ribose) polymerase (PARP) and apoptosis regulator (BAX) release and apoptotic index. Furthermore, the lead-treated group had marked increase in ROS, lipid-ROS, and Malondialdehyde (MDA) in comparison to the standard group. One more drawback of lead is that it diminish sirtuin (SIRT1) gene release, elevating the autophagy-related proteins level, including P62, ATG5, Beclin-1 and LC3 Ⅱ/Ⅰ. Because, resveratrol (40 μmol/L), an activator of SIRT1, it is able to restore the deleterious impacts of lead (200 μmol/L) to levels indistinguishable from standards (48).

On the other hand, Farideh Koohian et al., 2017 searched the radioprotectant consequence of resveratrol in opposition to genetic toxicity elicited by γ-irradiation in mice blood lymphocytes, as Ionizing radiation induces DNA damages similar to the genotoxic effect of lead which enhance the production of free radicals (49); Resveratrol as a nonpolar molecule has simple diffusion through cell membranes and immediately eliminate the free radicals produced as a result of lead exposure (50). Resveratrol increased hemeoxygenase I expression and extracellular Glutathione (GSH) (51). RSV is able to restore the levels of intracellular antioxidants such as GSH peroxidase, superoxide dismutase (SOD), and catalase (CAT) activity, bringing about neutralization of DNA damage by free radicals (52).

Conclusion

The results above showed that lead exposure leads to variable degenerative changes in the parotid salivary glands of adult male albino rats. Resveratrol showed histologically, immunohistochemically and by measuring DNA damage its efficacy in regenerating the parotid glands in rats. However, further clinical and histological studies with different parameters should be performed to evaluate the effect of long-term treatment use of resveratrol as a prophylaxis and therapeutic agent against lead toxicity. Besides, more complex studies should be done on biological and health consequences on human as lately, many compounds were examined as antioxidant agents, but harmful and proficiency for human application are continue to be a major concern.

Limitations of the study

Despite the safety of resveratrol, it still needs to be carefully investigated. Although the results of our animal experiments support the potential extrapolation of our hypothesis to human patients, species-specific differences in histology between humans and rats and differences in physiology, genetics, and disease manifestation between both species remain a limiting factor that can restrict the soundness of animal studies as predictors of human outcomes. Furthermore, variations in laboratory procedures, environmental conditions, and the way diseases are induced in animals can complicate translation.

Data availability statement

The datasets generated and/or analysed in this study are available from the corresponding author upon reasonable request.

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التأثير العلاجي المحتمل للريسفيراترول على الغدد النكفية المعرضة للرصاص في الجرذان البيضاء: نظرة ثاقبة على علم الأنسجة الغدية وتلف الحمض النووي

الملخص

الخلفية والأهداف: يُعدّ تعرّض الجمهور للرصاص خطرًا على الصحة نظرًا لآثاره الضارة طويلة الأمد. ونظرًا لنجاح استخدام مضادات الأكسدة كخيار علاجي، فقد اقتُرح استخدام الريسفيراترول للوقاية من الآثار الضارة الناجمة عن التعرّض للرصاص. تهدف هذه الدراسة إلى فحص التغيرات النسيجية والمناعية النسيجية والحمض النووي التي قد تطرأ على الجهاز الأنبوبي العنيبي للغدد اللعابية النكفية لدى فئران ألبينو البالغة بعد تناولها أسيتات الرصاص، وتحديد التأثير الوقائي المحتمل للريسفيراترول. الطرق: شملت عينة الدراسة 45 فأراً ذكراً من سلالة ويستار. تلقت المجموعة الأولى 50 مل/كجم/يوم من الماء المقطر لمدة خمسة وخمسين يوماً . تلقت المجموعة الثانية 50 ملغ/كغ/يوم من أسيتات الرصاص لمدة خمسة وخمسين يوماً. تلقت المجموعة الثالثة 50 ملغ/كغ/يوم من أسيتات الرصاص لمدة 55 يوماً، بالإضافة إلى 10 ملغ/كغ/يوم من الريسفيراترول لمدة 28 يوماً. عند انتهاء الدراسة، تم تخدير الفئران، واستئصال غددها النكفية، وفحصها باستخدام صبغة الهيماتوكسيلين والإيوسين، واختبار الكاسبيز 3، واختبار المذنب. النتائج: كشف الفحص المجهري في المجموعة الثانية عن تغيرات متدهورة ملحوظة في الغدة النكفية. في المقابل، لوحظ في المجموعة الثالثة تجدد ملحوظ بعد حقن الريسفيراترول. أظهرت مجموعة الريسفيراترول انخفاضًا كبيرًا في تدهور الحمض النووي في الغدة النكفية، بما يتناسب مع مجموعة الرصاص، ويكاد يكون مساويًا للمجموعة الضابطة. الاستنتاج: أظهر الريسفيراترول فعالية ملحوظة في تجديد الغدد اللعابية النكفية للفئران المعرضة للرصاص.