Abstract

Background

Seasonal heavy metal pollution of river waters poses a significant risk to environmental and human health; yet its biological effects are often insufficiently characterized.

Aims

The aim of this study was to investigate the seasonal variations in river water pollution and to evaluate the associated cytotoxic, oxidative, and genotoxic effects of these waters on human keratinocytes using an integrated in vitro approach.

Methods

This study collected river water samples during the four seasons and evaluated their cytotoxic and genotoxic impacts in human keratinocytes using an integrated in vitro approach. The cells were exposed to the samples for 24 and 48 h. Cytotoxicity was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, elemental accumulation by inductively coupled plasma–mass spectrometry, and oxidative and genotoxic responses by assessing reactive oxygen species (ROS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels.

Results

All samples induced time- and season-dependent cytotoxicity, whereas control cells were unaffected. Autumn-collected samples exhibited the maximal cytotoxicity, with cell mortality ranging from 38.94% to 80.09% at 24 h and increasing to 56.97%–81.03% at 48 h. Spring-collected samples induced the second-highest lethality, with cell death reaching ≤90.03% at 48 h. Enhanced cytotoxicity was associated with elevated cellular levels of Fe, Al, Cr, Ni, Ca, and Sr at 24 h and Ti, Mn, Zn, Cu, and P at 48 h, indicating rapid uptake and delayed accumulation patterns. River water exposure also significantly increased ROS generation (p < 0.005) and 8-OHdG levels, particularly by autumn and spring samples, demonstrating cumulative oxidative DNA damage.

Conclusion

These findings indicate that seasonal river water pollution induces oxidative stress mediated cytotoxicity and genotoxicity in human keratinocytes. The study underscores the necessity of incorporating biological endpoints into routine water quality monitoring to better assess health risks associated with contaminated freshwater systems.

Introduction

Water pollution is a major environmental challenge driven by industrialization, agriculture, environmental factors, insufficient freshwater resources, and inadequate sewage treatment systems. Industrial activities constitute a dominant source of aquatic contamination, particularly effluents released from textile, food processing, pulp and paper, iron, and steel factories; distilleries and tanneries; and nuclear power plants (Chowdhary et al., 2019). These industries discharge wastewater containing volatile organic compounds, toxic chemicals, inorganic pollutants, and hazardous solvents, often without sufficient treatment. They also contaminate surface waters with heavy metals (HMs), the most prevalent being chromium, cadmium, and arsenic (Chen et al., 2019). For example, anthropogenic activities have resulted in extensive hexavalent chromium pollution in the central regions of the Loess Plateau, China (Ge et al., 2020). With accelerating urbanization, the volume and complexity of industrial wastewater discharges continue to increase (Wu et al., 2020).

Agricultural activities represent another significant contributor to water pollution, primarily through runoff containing organic waste, nitrogen-based fertilizers, and pesticides. Cropping systems release pesticides, soil sediments, nitrates, phosphates, salts, and pathogens into aquatic environments, degrading the ecosystem and posing potential risks to human health (Parris, 2011). Such pollution severely affects freshwater ecosystems (Moss, 2008) and poses additional concerns related to food safety and HM accumulation (Lu et al., 2015). In parallel, environmental factors can also influence water quality; elevated levels of trace elements, sodium, and salinity are associated with poor river water quality in regions such as the Loess Plateau (Xiao et al., 2019). In many developing countries, inadequate sewage treatment infrastructure and insufficient investment in water supply systems further exacerbate contamination-associated problems, enhancing the exposure to industrial chemicals, HMs, and algal toxins (Wu et al., 1999).

The impacts of contaminated water on human health are well documented, with polluted drinking and surface waters contributing to gastrointestinal, infectious, and chronic diseases worldwide. The use of water filtration systems or desalinated water in households markedly reduces the incidence of diarrheal diseases compared to untreated municipal water (Yassin et al., 2006). In contrast, the consumption of tap water is associated with an increase in gastrointestinal disorders (Payment et al., 1997). Beyond systemic health effects, recent research emphasizes that water pollution levels exhibit pronounced seasonal and spatial variability, which directly influences the concentrations of contaminants and associated risks. Hammoumi et al. (2024) reported significant seasonal quality fluctuations in the surface water of the Nador Canal; water quality was lower during the summer due to combined natural and anthropogenic factors, including agricultural runoff and industrial discharges. Similarly, in Anambra State, Nigeria, Amaechi et al. (2025) observed elevated levels of Cd, Pb, Zn, and Cu in river water during the dry season, primarily driven by industrial effluents, with a partial dilution of these contaminants occurring during the wet season. At the cell level, exposure to HM-contaminated water induces oxidative stress and inflammatory responses, leading to tissue damage and increasing susceptibility to chronic diseases. Kolawole et al. (2025) demonstrated that cadmium, lead, and arsenic induce a remarkable elevation in oxidative stress biomarkers and inflammatory mediators, underscoring a mechanistic link between environmental contamination and adverse biological outcomes. Water pollution is also associated with dermatological disorders, including melanosis, keratosis, hair loss, scabies, and skin cancer, particularly in populations using contaminated surface waters for drinking purposes. Chronic exposure to arsenic-contaminated water causes severe skin-related health effects (Kazi et al., 2009), while polluted rivers and industrial waters increase the incidence of scabies and skin cancer (Arif et al., 2020; Hanif et al., 2020). Meta-analyses further support the association between polluted aquatic environments and skin conditions such as erythema and pruritus (Fleisher & Kay, 2006; Yau et al., 2009).

Despite this extensive body of epidemiological and chemical evidence, experimental findings directly assessing the effects of river water samples on healthy human skin cells remain limited. A small number of in vitro studies have evaluated the biological toxicity of surface or river waters using aquatic organisms or nonhuman models. For instance, Amaechi et al. (2025) demonstrated season-dependent cytotoxic effects of river water samples on fish-derived cell lines, attributing toxicity to elevated HM loads, while genotoxic endpoints were not examined. Similarly, Hammoumi et al. (2024) assessed seasonal variations in surface water quality and reported reduced biological quality during summer months; however, their evaluation was restricted to physicochemical parameters without cell-based toxicity or DNA damage analyses. Overall, existing in vitro investigations have predominantly focused on nonhuman models, have been limited to single locations or seasons, and have largely assessed cytotoxicity alone, leaving genotoxic outcomes and human-relevant dermal exposure risks insufficiently characterized. To address this gap, the present study focuses on the Meriç–Ergene River Basin, a region extensively impacted by industrial discharges, agricultural runoff, and urbanization. Water samples were collected from nine strategically selected stations along the Meriç, Ergene, and Tunca rivers to capture spatial variability, hydrological characteristics, and pollution gradients. Using a healthy human keratinocyte line, we evaluated the cytotoxic effects (on cell viability) and genotoxic potential of the collected samples. By integrating spatially distributed environmental sampling with human-relevant in vitro assays, this study provides biomonitoring-based evidence that directly links surface water pollution to potential dermal health risks, extending beyond conventional ecological or chemical assessments.

Materials and Methods

Sampling Stations and Transport of Samples

Water samples were collected from three major rivers the Meriç (Maritsa), Ergene, and Tunca rivers located in northwestern Türkiye. The Meriç River forms part of the border between Türkiye and Greece and flows southward into the Aegean Sea, while the Ergene and Tunca rivers are important tributaries draining agricultural and industrial regions before discharging into the Meriç River. At each station, water from underneath the surface was collected, against the flow, and stored in 2 L brown glass bottles. Samples were transported to the laboratory under cold-chain conditions. The sampling station locations are detailed in Table 1. In situ measurements of the routine physicochemical parameters of the water at each sampling station, including temperature (℃), pH, and electrical conductivity (µS/cm), were performed at the time of collection with a portable multi-parameter water quality meter calibrated according to the manufacturer’s instructions. These parameters were recorded to characterize the prevalent environmental conditions at the time of sampling and support an interpretation of the findings of the subsequent biological and toxicological analyses.

Cell Culture

Human keratinocyte cell line (HaCaT) was cultured in Dulbecco’s Modified Eagle Medium: Ham’s F-12 medium supplemented with 1% L-glutamine, 5% fetal bovine serum, and 1% penicillin/streptomycin. The cells were placed in a cell culture incubator and cultivated at 37 ℃ under 5% CO₂ and 95% humidity. They were subcultured upon reaching an 80%–90% confluency.

Cytotoxicity Determination via 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) Assay

The cytotoxic impacts of river water on HaCaT cells were evaluated using the MTT assay. Cells were seeded in a 96-well plate at a density of 2 × 10⁴ cells/well in 180 µL of medium. After incubation for 24 h, the cells were exposed to river water applied at nine concentrations: 100%, 50%, 25%, 12.5%, 6.25%, 3%, 1.5%, 0.75%, and 0.39%, for 24 and 48 h. Then, 20 µL of 5 mg/mL MTT solution (in phosphate-buffered saline) was added to each well. All experiments were conducted in four replicates (n = 4). The plates were incubated for another 2 h, and the formazan crystals formed were dissolved in 100–200 µL of dimethyl sulfoxide. The OD₅₇₀ was measured, and cell viability and mortality were calculated by applying the formulae-formulas:

Viability (%) = (Experimental absorbance/Control absorbance) × 100

Mortality (%) = 100 – Viability (%)

Determination of Cell Elemental Concentrations Employing Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

HaCaT cells were seeded into 24-well culture plates at a density of 1 × 10⁵ cells/well and incubated at 37 ℃ for 24 h in a humidified atmosphere containing 5% CO₂. Following cell attachment, the culture medium was replaced with river water samples, to which the cells were exposed for 24 and 48 h. These durations were selected based on the cytotoxicity thresholds determined via the MTT assay to avoid excessive cell death. After exposure, the cells were harvested and acid digested for elemental analyses with 65% ultrapure HNO₃ (trace metal grade), using a controlled combustion/digestion procedure until complete mineralization. Subsequently, the digested samples were diluted to appropriate volumes with ultrapure water before analysis. Cell elemental concentrations were quantified using a 7700 series ICP-MS (Agilent Technologies, Inc., CA, USA). Calibration curves were plotted using certified multi-element standard reference (high-purity) solutions in the range of 0–1000 ppb prepared by serial dilution. Calibration standards were used at five concentrations, covering the samples’ expected range. Curve linearity was evaluated, and correlation coefficients (R²) were verified to ensure acceptable performance levels. Quality control samples were employed to verify calibration validity and signal consistency before proceeding with sample measurements. Calibration standards were measured periodically to ensure analytical accuracy and instrument stability. Data obtained only under stabilized and calibrated conditions were included in the final analysis. Instrument operating parameters, including plasma conditions, nebulizer settings, and acquisition modes, are detailed in Table 2. All samples were analyzed in triplicate (n = 3), and elemental levels were expressed as mean values. Statistical analyses were employed to identify elements with significant variations between experimental groups. Subsequently, principal component analysis (PCA) was applied to such elements to evaluate the patterns in elemental concentration variations and potential exposure duration-related clustering.

Assessment of Reactive Oxygen Species (ROS) Levels

HaCaT cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well and incubated for 24 hours at 37 ℃ and under 5% CO₂. Water samples at toxic dosages (ascertained utilizing the MTT assay) were applied to the cells, and incubated for 24–48 h. Experiments were performed in three replicates (n = 3). The ROS levels of these cells were measured spectrophotometrically with a commercial ROS detection kit (Shanghai Sunred Biological Technology Co., Ltd., Shanghai, China); the assay procedure is outlined in Table 3. Statistical significance (Sig.) was evaluated using analysis of variance (ANOVA), followed by Student’s t-test for pairwise comparisons. Differences between the mean ROS assay expression values obtained were assessed employing Student’s t-test.

Assessment of Oxidative DNA Damage using 8-Hydroxy-2′-Deoxyguanosine (8-OHdG) as a Biomarker

HaCaT cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well and incubated for 24 h at 37 ℃ under 5% CO₂. DNA was extracted from the cells after exposure to toxic doses of contaminated river water for 24 and 48 h. Oxidative DNA damage was assessed utilizing reactions catalyzed by Nuclease P and phosphatase. Oxidative stress-related markers were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Jet Stream 6460 Triple Quadrupole instrument (Agilent Technologies). The analysis parameters are provided in Table 4.

Statistical Analysis

Probit analysis was applied to the MTT assay results to determine the 50% lethal dose (LD₅₀), in accordance with the Turkish Standards Institute (TSE) and ISO 10993-5 guidelines. Prior to parametric analyses, the assumption of homogeneity of variances was assessed using Levene’s test. When variance homogeneity was satisfied (p > 0.05), parametric tests were applied. Comparisons involving more than two groups (e.g., seasonal or station-based differences in cytotoxicity, ROS production, elemental accumulation, and 8-OHdG levels) were evaluated using one-way ANOVA. When significant differences were detected, Tukey’s post hoc test was applied to identify pairwise group differences. In cases where the assumption of homogeneity of variances was violated, Welch’s ANOVA followed by the Games–Howell post hoc test was used. Comparisons between two groups were performed using Student’s t-test or Welch’s t-test, as appropriate. PCA was used to explore patterns in ICP-MS-derived elemental profiles. Correlations between 8-OHdG levels were assessed using Pearson’s or Spearman’s correlation analyses, depending on data distribution. Statistical analyses were performed using XLSTAT, GraphPad Prism, and SPSS v25.0. A p < 0.05 was considered statistically significant.

Results

Physicochemical Characteristics of the River Water Samples

The physicochemical properties of the river water samples, including temperature, pH, and electrical conductivity, measured at the time of sampling, are summarized in Table 5. Pronounced seasonal variations were observed in these parameters across sampling stations, with temperature being minimum during winter (8.0–12.0 ℃) and maximum during the summer (27.0–31.0 ℃). The pH values remained slightly alkaline, ranging from 7.36 to 8.33, throughout the study period, and exhibited only minor seasonal fluctuations. In contrast, electrical conductivity exhibited pronounced spatial variability, with substantially elevated values recorded at station E (2200–4300 µS/cm) and the least at stations M and T (339–923 µS/cm), likely reflecting differences in hydrological conditions and local anthropogenic influences among the three rivers. Overall, electrical conductivity values were higher during autumn and winter than in spring and summer. These baseline physicochemical characteristics provide essential contextual information for the interpretation of subsequent cell and toxicological findings.

Analysis of Cytotoxicity Results

The cytotoxicities of various river water samples were evaluated, and the findings are presented in Figure 1. The results demonstrated an obvious time-dependent increase in mortality rates among most treatment groups compared to the control (nonexposed) group, which exhibited 0% mortality. The asterisk (*) above the bars indicates statistically significant variations between the two groups. The TSE ISO 10993-5 standard considers a test material to be cytotoxic if it causes >30% cell mortality (<70% viability). A majority of the samples were classified as toxic, based on this guideline. Such cytotoxicity was most pronounced after 48 h of exposure and was particularly severe in the samples collected during Autumn (e.g., T1–T3, M1–M3, and E1–E3), with mortality rates consistently >50%. These findings confirm the marked presence of cytotoxic agents in river water, with the maximum toxicity induced by the Autumn samples at the 48 h time point (Figure 1 and Table 6).

Results of the Intracellular Element Levels Determination

This study provides evidence with robust statistical Sig. that the concentrations of trace elements within cells exposed to river water are highly sensitive to sampling location and exposure duration. PCA demonstrated that, at 24 h and 48 h of exposure, a majority of the total variance was explained by the first PC1, indicating that dataset variability was primarily driven by differences in element accumulation levels among exposure groups. PCA further revealed robust positive correlations among Fe, Al, Ca, Cr, and Ni at 24 h and among Ti, Mn, and P at 48 h. These observations suggest that such elements either originate from a common environmental source or are taken up by cells through shared or closely related biochemical pathways. Moreover, a clear and statistically significant separation of the E group from all others (prominent main effect, p < 0.001) confirms that source-specific differences in river water composition are the primary determinants of variations in intracellular element accumulation levels. Extension of exposure duration from 24 to 48 h identified two distinct accumulation dynamics. The absence of statistically significant variations in Fe, Al, Cr, and Ni contents at 24 and 48 h indicates rapid intracellular saturation or cell-regulatory mechanisms achieving equilibrium within the initial 24 h of exposure. In contrast, the Ti, Mn, Zn, Cu, and P contents elevated significantly at 48 h, suggesting that these elements may require prolonged periods of exposure for accumulation or follow slower uptake kinetics. A particularly crucial finding was the pronounced time–season interaction observed with Ca and Sr. Their concentrations increased markedly from 24 to 48 h, with those of Ca and Sr elevating by 1.7–2.58-fold and 1.17–2.10-fold, respectively. Such variability depended on the season, indicating that Ca and Sr accumulation was remarkably influenced by exposure duration and environmental conditions, positioning these elements as plausible, high-sensitivity biological indicators of seasonal and environmental dynamics. In conclusion, the findings of the combined PCA and variance analyses demonstrate that the river water environments investigated were heterogeneous regarding trace element composition and that cell responses differed prominently depending on the water source. These findings underscore the critical nature of including exposure duration and seasonal variability in environmental pollution risk assessments and toxicological evaluations (Figure 2).

ROS-Mediated Oxidative Stress and DNA Damage Assessed by Measuring 8-OHdG Levels

The differences in ROS levels within the HaCaT cells exposed or not exposed to river water for 24 and 48 h were evaluated using the independent samples t-test, preceded by Levene’s test for equal variances. As most groups showed two-tailed Sig. values >0.05, the assumption of equal variances was generally met. The t-test results, specifically the Sig. values, revealed that ROS levels of most treated group cells differed with high Sig.compared to the control group ones (Sig. 0.005). The variations among most samples from the E, M, and T ecosystems across seasons were statistically significant (*). ROS levels induced by only three spring samples, E2-Spring (Sig. = 0.695), M1-Spring (Sig. = 0.251), and T3-Spring (Sig. = 0.070) did not differ with statistical Sig. from the contents of control group cells at the tested time points. These trends suggest that spring-collected samples induced a negligible oxidative stress response in HaCaT cells (Figure 3, Table 7).

As expected, control group cells demonstrated the lowest 8-OHdG levels at both 24 and 48 h of treatment, indicating minimal DNA damage under normal cell culture conditions. In contrast, cells of all groups exposed to river water exhibited markedly elevated 8-OHdG levels compared to the control group cells. Such an increase confirms that pollutants present in river water induced oxidative stress and consequent DNA damage in the HaCaT cells.

The overall trend indicated an increase in DNA damage with prolonged pollutant exposure. As illustrated, cell 8-OHdG levels were higher at 48 h compared to 24 h upon treatment with water collected from several locations, e.g., E1-Winter, E2-Winter, M1-Winter, M2-Spring, and T2-Winter. However, DNA damage induced by water collected from a few locations declined at 48 h relative to 24 h (e.g., E1-Autumn). This observation suggests activation of DNA repair mechanisms or extensive cell death at 48 h, which reduced the number of viable cells or DNA available for measurement. The T2-Spring group cells exhibited the maximal 8-OHdG levels at 48 h (~18), indicating the most severe DNA damage and suggesting that the pollutants at this location, as well as the collection season exerted the strongest genotoxicity. Conversely, the E1-Autumn group cells showed the highest 8-OHdG contents at 24 h (~ 13.5), implying a rapid and potent induction of oxidative stress by river water during autumn. In winter group cells, particularly the M2-Winter and T2-Winter group ones, a pronounced elevation in 8-OHdG levels was detected at 48 h (Figure 4). Overall, the severity and duration of oxidative stress–induced DNA damage may vary remarkably depending on exposure time generally increasing at 48 h as well as on the river ecosystem type and seasonal conditions.

Discussion

The present study evaluated the toxicological effects of river water with seasonal variations in HM pollution levels. Cytotoxicity was assessed in the HaCaT human keratinocyte model to assess potential relevance to human health. The pronounced exposure time- and sampling season-dependent impacts observed indicate that river water-induced toxicity is highly dynamic and robustly influenced by seasonal variations in environmental conditions. Similar to previous reports demonstrating region-specific HM accumulation patterns in aquatic ecosystems (Praveen et al., 2016; Shetaia et al., 2023), the findings of the present study suggest that seasonal fluctuations in contaminant inputs critically shape responses in biological systems.

The elevated cytotoxicity induced by autumn- and spring-collected samples is likely linked to elevations in the pollutant loads of river systems caused by surface runoff and leaching. During these periods, fertilizers, pesticides, and industrial residues are well-recognized contributors to freshwater contamination, and their biological relevance is reflected in the substantial accumulation of multiple elements within cells detected by ICP-MS. Rapid uptake of Fe, Al, Cr, Ni, Ca, and Sr within the first 24 h suggests an early cell response to environmental exposure. In contrast, the delayed accumulation of Ti, Mn, Zn, Cu, and P at 48 h underscores the amplification in toxic effects induced by prolonged exposure.

Among the elements analyzed, Ca and Sr displayed a particularly pronounced time–season interaction. Given the central role of Ca in cell signaling, mitochondrial regulation, and membrane stability, a dysregulation in intracellular Ca accumulation may intensify cytotoxicity-associated pathways. The parallel increase in Sr, which is chemically similar to Ca, may further interfere with Ca-dependent cellular processes. These observations support the notion that Ca and Sr may function as sensitive cell-level indicators of the influence of seasonal environmental variability and pollutant pressure.

The biological relevance of HM accumulation was further substantiated by oxidative stress and genotoxicity endpoints. Significantly elevated ROS levels in most exposed cell groups indicate that oxidative stress represents a key mechanism underlying the observed cytotoxicity. Only a limited number of spring samples failed to induce a marked elevation in ROS production, consistent with their comparatively lower toxic profiles. Increased ROS generation was accompanied by an elevation in 8-OHdG levels, reflecting oxidative DNA damage and cumulative genotoxic impacts with prolonged exposure. These findings align with established evidence indicating that HMs exert genotoxicity through ROS-mediated mechanisms and the disruption of DNA repair pathways (Jadoon & Malik, 2017).

Exposure to HMs such as cadmium, lead, and arsenic results in oxidative stress, mitochondrial dysfunction, and apoptosis in human cells, including keratinocytes (Davodpour et al., 2019; Habibi et al., 2022; Li et al., 2022; Mohamed et al., 2024; Sobhanardakani, 2017, 2019).

Moreover, elevated contents of oxidative DNA damage markers, including 8-OHdG, have been detected in individuals exposed to contaminated water or HMs (Mohod & Dhote, 2013; Szymańska-Chabowska et al., 2009). The results of the present study are therefore consistent with existing toxicological evidence linking exposure to metal-contaminated environments and adverse human health outcomes.

The genotoxic potential of HMs observed in this study is further supported by similar findings made in plant and aquatic organism models, where exposure to HM-contaminated waters has been associated with chromosomal aberrations, micronuclei formation, and DNA fragmentation (Cavusoglu et al., 2010; Chatterjee & Chatterjee, 2000; Doğan et al., 2022; Scalon et al., 2010). Such cross-species consistency reinforces the ecological and biological relevance of the oxidative and genotoxic responses detected in HaCaT cells.

Although cadmium has been recognized as the predominant ecological threat in certain aquatic environments (Shetaia et al., 2023), the present study identified a region-specific contamination profile dominated by Fe, Al, Cr, and Ni. Such a discrepancy highlights that toxicity induced by polluted freshwater cannot be generalized across regions and underscores the necessity of site-specific assessments that consider local anthropogenic activities and environmental conditions.

Overall, the present findings demonstrate that seasonal variations in river water pollution exert significant cytotoxic and genotoxic impacts through oxidative stress-mediated mechanisms. Rather than reiterating contamination levels, these results emphasize the broader implications of sustained and seasonally-modulated pollutant exposure for human health and ecosystem integrity. Consistent with previous calls for comprehensive monitoring of metal-contaminated aquatic systems (Shetaia et al., 2023), this study underscores the importance of continuous, region-specific monitoring strategies and targeted mitigation policies addressing both agricultural and industrial sources of pollution.

Conclusion

This study comprehensively investigates the toxicity levels of river waters sampled at various locations, focusing on their impacts on human skin cells at the physiological and molecular levels. The findings offer valuable insights into the harmful effects of water pollution on human health, specifically highlighting the toxicological risks posed by HM-contaminated water. Our research demonstrates that exposure to HM-polluted river water induces significant levels of oxidative stress, DNA damage, and genotoxicity in human skin cells. Moreover, these effects varied in extent across seasons and exposure durations, underscoring the dynamic nature of water contamination and its impact on biological systems. Among the HMs analyzed, Al, Fe, and Cr were identified as key inducers of DNA damage, suggesting that these metals may serve as potential biomarkers for assessing toxicity in human skin cells. The study emphasizes the need for continuous water quality monitoring, especially in regions affected by industrial and agricultural pollutants, and advocates for further research into the mechanisms underlying HM-induced toxicity. These findings are crucial for developing effective strategies to mitigate the health risks associated with contaminated water and for advancing the protection of public health in the face of growing environmental challenges.