The main goal of this investigation was to determine the incidence of nuclear anomalies, including micronuclei, in mucosal cells exfoliated from cancer patients.

The objective of this study was to quantify DNA damage in cancer patients and identify the presence of micronuclei and other nuclear abnormalities in exfoliated buccal cells of the mouth by combining hematologic and biochemical monitoring with a comet assay.

Whole blood samples were collected from both cancer patients and the control group between 2/12/2023 and 20/5/2024. Approximately 5 mL of venous blood was drawn from each participant using sterile disposable syringes. The collected blood was immediately divided into two portions:

Portion 1: Transferred into EDTA-coated tubes for immediate analysis of hematological indices. A part of this portion was also processed directly for the Comet assay (Single Cell Gel Electrophoresis) and stored at −20°C for future Comet assay analysis.

Portion 2: Transferred into heparinized tubes for biochemical analysis, which was performed directly on the same day of collection.

All samples were maintained at 4°C in a cooling box during transportation and were processed within 4 hours of collection to ensure cell viability and prevent DNA degradation. Samples designated for the Comet assay that were not processed immediately were stored at −20°C until analysis.

A total of 190 samples (45–60 age) were collected from random people with and without cancer, 90 from healthy people (without cancer), and 100 from people with different types of cancer (prostate cancer, breast cancer, colon cancer, lymphatic gland cancer, and lung cancer). Histopathological diagnoses were confirmed by the oncology center for each patient. Sample were collected from buccal cells for micronucleus assay, and the blood for comet assay, and other biological indices.

The investigation was conducted as follows, following the steps described by Gopal and Padma¹⁴: following the instructions for washing their salivary glands with water, the participants collected cells from the mucous membrane of their mouths using a wooden spatula that had been previously moistened, and subsequently placed on a clean microscope slide. They were air-dried and preserved in methyl alcohol (96%) for 3–5 min. The specimen was stained with May-Grunwald stain for 20–25 min, followed by Giemsa staining for 3–5 min, wished, allowed to dry, and examined under a microscope (40x). The incidence of micronuclei was documented, with 2000 cells assessed for each subject.

During this test, a treatment technique (Trevigen, Inc., Gaithersburg, US) was applied. The Lysis Solution was prepared and then allowed to cool for 20 min. Add cells with 1 × 10⁵/ml melted low point of melting (LMA) after 50 μl is dispensed onto a Comet SlideTM. Agarose. Agarose and cells were evenly distributed throughout the sample using a pipette tip. After being flattened, the microscope slides were placed in the dark environment and cooled for 10 min at 4°C. To improve sample adherence in heavy humidity, the gelling time might be prolonged to 30 min. After 30 to 60 min, the slides were removed out of a 4°C Lysis Solution to get rid of any remaining buffer that Com-. For one hour, a freshly made Alkaline unwinding Solution from the company—which had a pH higher than 13—was submerged at 4°C. The slides were placed in a tray with labels toward the black cathode after the alkaline electrophoresis solutions were added. The power was switched on for 30 min at a voltage of 21 volts. Electrophoresis was performed at 21 V for 30 min; the field strength was calculated as 21 V divided by the distance between the electrodes (cm). The samples were carefully immersed twice in 70% ethanol for five minutes, twice in distilled water, and twice in electrophoresis solution. The materials were examined after being dehydration at 37°C for 10 to 15 minutes. After applying 1X Ethidium Bromide to the Comet Slide TM slide to eliminate discoloration, cold distilled water was used for rinsing. The slides were quickly assessed. A fluorescent microscope with a 40X lens plus a touchscreen camera enabling slide processing was used to measure the DNA damage. Four comet images were evaluated using 50 randomly selected cells, accounting for 25 cells per plate. We examined the number and rate of migration of the damaged cells.¹⁵

Urea, creatinine, calcium, and total serum bilirubin levels were estimated using the COBAS C111 device, while the Converges device was used for complete blood count.

A t-test was used to analyze the data. Duncan’s original varied range test was used to minimize the mean difference as the mean ± SD. SPSS (version 28.0, SPSS Ltd., Surrey, UK) was used for each statistical analysis.

Ethical Approval: This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. The research protocol was reviewed and approved by the Pharmaceutical Research Ethics Committee (PREC) at the University of Mosul, College of Pharmacy (Reference Code: PREC-25-3-10; Date: October 12, 2025).

Note: The title mentioned in the ethical certificate [Ref: PREC-25-3-10] represents the initial study title; the current manuscript title is an updated version of the same study.

Informed Consent: Written informed consent was obtained from all individual participants included in the study. All participants were fully briefed on the study’s objectives, the blood collection procedure, and their right to withdraw at any time. Confidentiality of all personal and medical data was strictly maintained throughout the study.

Identifying indications of early biological impacts is the aim of examining micronuclei in buccal epithelial cells. The frequency of micronuclei in buccal epithelial cells was determined by producing violet-colored samples and examining the outcomes under a 40X magnification microscope. As shown in Table 1, all micronucleus parameters were significantly higher in cancer patients compared to the control group (p < 0.001).

The quantitative comet assay parameters for both groups are presented in Table 2. The mean % Tail DNA was significantly higher in cancer patients (55.27 ± 38.13) compared to healthy controls (8.94 ± 6.56), with a highly significant difference (p < 0.001). Similarly, the Olive Tail Moment was significantly elevated in the patient group (48.57 ± 44.89) versus controls (2.11 ± 1.80), p = 0.001. The Tail Length was also significantly greater in cancer patients (27.46 ± 5.90 px) compared to controls (6.31 ± 4.87 px), p < 0.001. These findings indicate substantially higher DNA fragmentation in cancer patients, confirming the genotoxic effects associated with malignancy.

As shown in Table 3 show a significant increase in the mean levels of urea (50.600 ± 8.259) and creatinine (1.073 ± 0.173), and no significant increase in TSB (0.917±0.143) and HCT (38.080 ± 2.311) in cancer patients compared with the control. Calcium (9.010 ± 0.680), Hb (8.322 ± 1.247), Plt (91.300 ± 24.495), and White Blood Cell count (11.920 ± 2.833) levels were significantly lower in the patient group compared to the control group.

The comet assay has been used extensively in genotoxicity investigations, bio-monitoring, ecological testing, and human illness research to quantify various cellular responses to DNA damage.²⁹ In addition to describing the assay’s utility in evaluating oxidative stress within tumors, its potential as a tool for predicting a person’s tumor sensitivity to radiation and other chemotherapeutic medications was investigated.

A comet assay, which involves single-cell electrophoresis on gels, was used to evaluate the extent of DNA impairment in exfoliated buccal cells. A DNA-binding dye was used to observe the results under a fluorescence microscope, displaying unique comet-like patterns that indicate the level of genotoxic stress experienced by each individual cell, as illustrated in Figure 2.

The image shows a range of comet morphologies among the studied cells, from intact nuclei with little tail production to those with prominent comet tails. These tails are a sign of DNA strand breaks, because they are created when fragmented DNA migrates during electrophoresis. Heterogeneous levels of DNA damage within the cell populations are suggested by differences in tail length and intensity.

Cells exhibiting long, diffuse tails with reduced head intensity were considered to have experienced substantial DNA disintegration, which is in line with the genotoxic insult frequently observed in cancerous cells. In contrast, cells with tiny or absent tails were thought to have sustained little damage, possibly indicating more resilient or less damaged subpopulations.²⁹

According to experimental circumstances, this damage pattern could be a result of the cytotoxic effects of external treatments (such as radiation, chemotherapy, or oxidative stress) or the inherent instability of epithelial tissue cancer cells. The results validate the usefulness of the comet assay as a quick and sensitive technique for tracking genotoxic reactions in vitro and identifying Breakage of DNA strands in individual cells.^30–32

These results were in agreement with those of Pullakanam et al.,³³ who showed a decrease in total white blood cells, hemoglobin, and platelets and an increase in HCT in cancer patients compared with controls. Additionally, it concurs with Rimsha et al.,³⁴ which showed significant (P < 0.05) variations among cancer patients and control groups in biochemical variables (ALT, AST, and cholesterol) and blood test results (RBCs, H.B., PCV, WBCs, and platelets).

Anemia, a common complication among cancer patients, is frequently detected by measuring hemoglobin and PCV levels; those that, when compared with healthy controls, were typically lower in the sick group.³⁵ Furthermore, these levels are significantly affected by factors including overall clinical health and dietary status.³⁶ Compared to controls, patients’ average corpuscular size and median concentration of corpuscular hemoglobin were significantly lower.

The discovery that kidney function was diminished in patients with cancer supported the findings of Ibraheem et al.,^37,38 who discovered that the levels of serum electrolytes and all kidney function tests were beginning at the upper limit or below the normal range, despite the fact that some levels showed significant variations among patients as well as controls.

Patients with cancer might have other health issues or characteristics that increase the possibility of renal impairment before receiving kidney-toxic therapy.³⁹ Chronic renal disease is prevalent in the elderly, regardless of the presence of cancer. The higher urea levels in the cancer group than in the healthy group may be due to increased protein metabolism following chemotherapy-induced cell death. An independent t-test was performed to compare biochemical and hematological parameters between patients and controls. The results, shown in Table 3, indicate statistically significant differences for most parameters, with patients exhibiting higher or lower values than controls. The mean levels of these parameters are visually represented in Figure 3, which illustrates the differences between the studied groups.

One limitation of this study is the absence of multivariate analysis to adjust for confounding factors such as age and smoking, due to the limited sample size. Future studies with larger cohorts are needed to confirm these findings.

The data supporting the findings of this study are openly available in the Zenodo repository at https://zenodo.org/records/18889026, reference number DOI: 10.5281/zenodo.18889026.⁴⁰

This dataset contains the SPSS statistical analysis outputs used in the manuscript, including descriptive statistics, tests of homogeneity of variances, ANOVA results, and means plots for the following variables: Urea, Creatinine, Ca, TSB, Hb, WBC, Hct, and plt. The data are aggregated to protect participant confidentiality and do not contain any individual patient information, in accordance with institutional ethics committee guidelines.

The dataset is published under the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

The authors would like to thank the staff of Salahaddin oncology centre for their support in sample collection and technical assistance.

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