Curcumin Attenuates Aroclor 1254‐Induced Oxidative, Genotoxic, and Apoptotic Alterations in HepG2 Cells

ABSTRACT

Polychlorinated biphenyls (PCBs), particularly Aroclor 1254 (A1254), are persistent environmental contaminants with hepatotoxic, genotoxic, and pro‐oxidant properties. The liver, as the main organ for xenobiotic metabolism, is highly vulnerable to PCB‐induced oxidative stress and DNA damage. Curcumin, a polyphenolic compound from Curcuma longa, exhibits potent antioxidant, anti‐inflammatory, and cytoprotective effects. This study investigated the oxidative, genotoxic, apoptotic, and mutagenic effects of A1254 in HepG2 cells and evaluated curcumin's protective potential. Cells were exposed to sub‐cytotoxic concentrations of A1254 (2.5–25 µM), curcumin (0.5–2 µM), and their combinations. ROS levels, antioxidant enzyme activities, oxidative biomarkers, and total antioxidant capacity were measured. DNA damage was assessed via 8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG), 8‐oxoguanine glycosylase (OGG1), and Comet assays. Apoptosis was evaluated by caspase‐3 and −8 activities, and mutagenicity via Ames (TA98/TA100) and HPRT assays. A1254 disrupted redox homeostasis, elevating ROS and oxidative damage while depleting glutathione, thiols, and enzymatic antioxidants. Curcumin co‐treatment restored antioxidant defenses and mitigated oxidative damage. Genotoxicity induced by A1254, indicated by increased 8‐OHdG and DNA strand breaks, was markedly reduced by curcumin, which also attenuated apoptotic responses. These results demonstrate that A1254 exerts multifaceted cytotoxicity via oxidative stress, DNA damage, apoptosis, and mutagenicity, and that curcumin effectively ameliorates these effects, supporting its potential as a hepatoprotective and genoprotective agent against PCB‐induced toxicity.

In HepG2 cells, Aroclor 1254 induced oxidative stress, DNA damage, apoptosis, and mutagenicity; co‐treatment with curcumin restored antioxidant defenses (GSH/thiols; SOD, CAT, GPx, GR, TrxR), reduced ROS and comet/8‐OHdG readouts, and attenuated caspase activation, demonstrating curcumin's hepatoprotective and genoprotective modulation of PCB toxicity.

1. Introduction

Polychlorinated biphenyls (PCBs) are organo‐halogenated compounds that were widely used for approximately 50 years as insulating agents, lubricants, and industrial additives due to their excellent electrical insulation and thermal resistance properties. In earlier decades, PCBs were widely applied to minimize the fire hazards of dielectric fluids and to increase the stability of elastic sealants. However, they are currently recognized as significant environmental contaminants and classified among the most critical persistent organic pollutants [1].

It is estimated that over one million tons of PCBs were produced worldwide until 1980, with approximately one‐quarter to one‐third of this amount released into the environment. Following two accidental contamination incidents in 1977 that resulted in human fatalities and serious health effects, the production of PCBs was banned [2, 3]. However, due to their high resistance to environmental degradation and strong bioaccumulation potential, these compounds continue to persist in the environment and in biological tissues, exerting toxic effects on various biological systems, particularly on the endocrine system [3, 4].

Exposure to PCBs in humans, experimental animals, and wildlife can occur through inhalation of contaminated air, ingestion of polluted food or water, as well as dermal or ocular absorption. The environmental persistence of PCBs, combined with their biomagnification through the food chain, represents a long‐term ecotoxicological risk on a global [5, 6]. Even though PCBs have been banned for many years, residual contamination at low concentrations continues to persist in the environment and within biological systems [7].

Aroclor 1254 (A1254) is a prominent commercial PCB mixture, distinguished by a chlorination level of approximately 54% by weight. The primary mechanism of PCB toxicity involves their binding to aryl hydrocarbon receptors (AhR), which triggers a cascade leading to the excessive production of reactive oxygen species (ROS). This oxidative stress, in turn, can induce severe cellular damage, including DNA damage, lipid peroxidation, and protein oxidation. Known adverse health effects of PCBs are broad, encompassing hepatotoxicity, immunotoxicity, reproductive toxicity, neurotoxicity, and endocrine disorders [8, 9, 10].

Strong evidence from large‐scale population studies has demonstrated a clear link between PCB exposure and liver diseases. The liver is highly susceptible to the toxic effects of PCBs due to its crucial role in detoxification processes [11]. Hepatotoxicity is caused by a variety of mechanisms, including rupture of the cell membranes, immune response induction, modification of the drug metabolic pathways within cells, increases in ROS, lipid peroxidation, apoptosis, and inflammation [12, 13]. Excessive formation of ROS in liver cells or a decline in the antioxidant defense capacity led to oxidative stress [14]. An excessive number of oxidative mediators can interact with proteins and lipids, causing oxidation of these biomolecules [12]. In addition to proteins and lipids, ROS can interact with DNA and cause genotoxicity. Various animal models and cell‐based test systems have been utilized to examine the genotoxic effects of industrial PCB mixtures. High doses of different PCBs (e.g., Aroclor 1254, 1221, and 1260) were found to cause DNA adducts, strand breakage, uncontrolled DNA synthesis, and chromosomal abnormalities in mammalian cells, fish, and transgenic mice [15].

Curcumin, the principal curcuminoid derived from Curcuma longa (turmeric), is recognized as the primary bioactive component responsible for the plant's diverse pharmacological properties. It exhibits strong anti‐oxidant and anti‐inflammatory activities and has been shown to influence multiple cellular signaling pathways [16, 17]. Evidences obtained from the in vivo and in vitro studies in the literature show that curcumin has hepatoprotective antioxidant activities via upregulating nuclear factor erythroid 2‐related factor 2 (Nrf‐2), increasing levels of glutathione (GSH) levels, and activities of antioxidant enzymes just as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT) [18, 19, 20]. In addition, curcumin shows a prominent ameliorative effect against different chemical and heavy metal‐induced genotoxicity [21, 22, 23].

Even though PCBs exhibit a wide spectrum of biological and toxicological characteristics, more research has to be performed to completely understand PCBs potential mechanism underlying the hepatotoxicity. In this study, we investigated the oxidative stress parameters and genotoxicity as the molecular mechanisms underlying Aroclor 1254‐induced hepatotoxicity and antioxidant curcumin's hepatoprotective effect against this particular PCB congener.

2. Materials and Methods

2.1. Chemicals and Kits

HepG2 cells (ATCC HB‐8065) were purchased from American Type Culture Collection (ATCC) (Rockville, MD). Intracellular ROS Kit, Aroclor 1254 (99%), curcumin, alcohols, paraffin, Tris, diethylenetriaminepentaacetic acid (DTPA), 3‐(4,5‐dimethylthiazol‐2‐yl)−2,5‐diphenyltetrazolium bromide (MTT), phenylmethanesulfonyl fluoride (PMSF), thiobarbituric acid (TBA), 1,1,3,3 tetraethoxypropane (TEP), glutathione reductase (GR), GSH, nicotinamide adenine nucleotide phosphate (NADPH), dimethyl sulphoxide (DMSO), methanol, potassium dihydrogen phosphate, potassium hydroxide, protease inhibitor cocktail and bovine serum albumin (BSA) were obtained from Sigma‐Aldrich (St. Louis, MO). Merck Masson's trichrome (M‐S), Mayer's hematoxylin solution, phosphate‐buffered saline (PBS), Periodic acid‐Schiff (PAS) kit, hydrogen peroxide (H₂O₂), pyrogallol, and n‐butanol were purchased from Merck (Darmstadt, Germany). Glutathione peroxidase (GPx1), catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (TAOC), thiobarbituric acid reactive substances (TBARS), protein oxidation, and Thiol Detection kits were obtained from Cayman Chemical Company (Ann Arbor, MI). Thioredoxin reductase (TrxR) assay Kit and GSH Assay Kit were purchased from Sigma‐Aldrich (St. Louis, MO). “Pierce BCA protein assay kit” was from Thermo Fisher Scientific (Waltham, MA). Dulbecco's phosphate buffer saline (DPBS), Dulbecco's modified Eagle's Medium (DMEM), fetal bovine serum (FBS), and penicillin G/streptomycin were purchased from Biowest (Riverside, MO). DNA Isolation Kit (Quick‐DNA Miniprep Kit) was from Zymo Research. 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) ELISA Kit was from OXISResearch (OXIS Health Products Inc., Portland, OR, USA). 8‐Oxoguanine glycosylase (OGG1) kit was purchased from MyBiosource (San Diego, CA). Caspase 3 and caspase 8 kits were obtained from Sigma–Aldrich (St. Louis, MO). Ames bacterial reverse mutation kit (Ames MPF™, microplate format; Salmonella typhimurium TA98 and TA100, with and without metabolic activation), including rat‐liver S9 mix, NADPH‐generating cofactor solution, and standard positive controls (2‐nitrofluorene, 4‐nitroquinoline‐1‐oxide, 2‐aminoanthracene) was obtained from Xenometrix AG (Allschwil, Switzerland). For mammalian gene mutation testing, materials for the CHO/HPRT forward mutation assay (OECD TG 476)—including 6‐thioguanine (6‐TG; selection agent) and HAT/HT supplements (hypoxanthine‐aminopterin‐thymidine for cleanup; hypoxanthine‐thymidine for post‐treatment recovery), Ham's F‐12 basal medium, l‐glutamine, sodium pyruvate, and 0.25% trypsin‐EDTA—were obtained from Thermo Fisher (Gibco) and Sigma‐Aldrich; ethyl methanesulfonate (EMS; –S9) and cyclophosphamide (CPA; +S9) were used as reference mutagens, and a rat‐liver S9 mix with cofactors (Sigma‐Aldrich) was used when metabolic activation was required.

2.2. Cell Culture

HepG2 cells (ATCC HB‐8065) after 4th passage were cultured in DMEM (supplemented with 10% FBS and 1% penicillin G‐streptomycin and were kept at 37°C and 5% CO₂ in an incubator (Heraeus Instruments, Hanau, Germany). After they reached 80% confluency, HepG2 cells were trypsinized, centrifuged at 1500xg for 5 min, and then sub‐cultured.

2.3. Preparation of A1254 and Curcumin

A1254 was dissolved in fresh sterilized peanut oil and then diluted in ethanol as a stock solution. Curcumin was dissolved in DMSO (0.1%) and then further diluted with cell culture medium.

2.4. Cell Viability

The cytotoxic effects of A1254 and curcumin were determined by MTT assay. Briefly, HepG2 cells were seeded in 96‐well plates at a density of 10⁴ cells/per well. The cells were incubated for 24 h in an incubator at 37°C, 5% CO₂. After the first incubation, cells were treated with different concentrations of A1254 (0, 2.5, 5, 7.5, 10, 12.5, 25 µM) and curcumin (0, 0.5, 1, 1.5, 2 µM) in cell medium for 24 h. Cell viability was assessed using the MTT assay (1 mg/mL). Briefly, 100 µl of MTT solution was added to each well, followed by incubation at 37°C for 3 h. Subsequently, the supernatant was removed, and 150 µL of DMSO was added to solubilize the formazan crystals. Absorbance was recorded at 570 nm using a SpectraMax M2 spectrophotometer (Molecular Devices, Sunnyvale, CA). The assay was performed in triplicate on three independent days, with two technical replicates per experiment. The data represent the mean values of all experiments. The control cells were expected to have 100% cell viability. The mean percentage of cell viability compared to the control group was used to express the results.

2.5. Experimental Groups

Cells were assigned to four groups:

• 1.Control (C): Untreated cells in complete medium; vehicle was matched across all groups [final DMSO ≤ 0.1% (v/v)].
• 2. Curcumin: Cells exposed to curcumin at 5 µM for 24 h (solubilized in DMSO; final DMSO ≤ 0.1% v/v).
• 3.Aroclor 1254 (A1254): Cells exposed to Aroclor 1254 at 38.7646 µM for 24 h.
• 4.A1254 + Curcumin (co‐treatment): Simultaneous exposure to Aroclor 1254 (38.7646 µM) and curcumin (5 µM) for 24 h.

Aroclor 1254 stock solution (10 mM) was prepared by dissolving 3.6 mg Aroclor 1254 in 1 mL sterile DMSO (v/v) and later diluted with medium with a final DMSO concentration of ≤ 0.1% (v/v). For the MTT assay, 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 20, 30, 40, and 50 µM concentrations were used. The inhibitory concentration 50 (IC₅₀) dose for Aroclor 1254 was found to be 26.78 µM. The inhibitory concentration 30 (IC₃₀) value (38.7646 µM) was used throughout the experiments.

Curcumin was prepared from a 10 mM stock [by dissolving 3.684 mg curcumin in 1 mL DMSO and later diluted with medium with a final DMSO concentration of ≤ 0.1% (v/v)]. For the MTT assay, 0, 0.5, 1, 2, 3, 5, 10, 20, 40, 50, 80, and 100 µM concentrations were used. After a careful consideration, 5 µM dose, which provided 100.04% cell viability, was used for protection studies.

These MTT experiments were therefore used as preliminary dose‐finding assays to identify one submaximally toxic Aroclor 1254 concentration (IC₃₀) and one non‐cytotoxic curcumin concentration (5 µM) for all subsequent mechanistic analyses, rather than to construct an extensive matrix of combination doses. All working solutions were prepared to keep the same final vehicle content across groups. Doses and exposure durations are provided in the corresponding assay subsections; concentrations were selected based on preliminary MTT results.

2.6. Intracellular ROS Production

Intracellular ROS levels were assessed using a commercial detection kit, which relies on the conversion of the non‐fluorescent probe 5‐(and 6‐) chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate into the green‐fluorescent derivative chloromethyl‐2′,7′‐dichlorofluorescein (CM‐DCF) within viable cells. ROS interact with the cytoplasmic fluorogenic substrate, generating a fluorescent signal proportional to the intracellular ROS concentration [24]. HepG2 cells (1 × 10³) were briefly washed with 90 µL of phosphate‐buffered saline (PBS). The ROS detection reagent stock solution was prepared by dissolving 40 µL of DMSO in the ROS detection reagent. Cells were treated with 10 µL of test compounds at their respective IC₃₀ concentrations, while control wells received 10 µL of PBS. To facilitate ROS induction, the 96‐well culture plate was incubated at 37°C in a 5% CO₂ atmosphere for 30 min. The master reaction mixture was prepared by combining 20 µL of the stock solution with 10 µL of assay buffer, and 100 µL of this mixture was immediately added to each well. Plates were then incubated for an additional 30 min under the same conditions. Fluorescence intensity was measured (λ _ex = 640 nm, λ _em = 675 nm) by using a spectrofluorometer (Spectramax M2, Molecular Devices). Control cells were expected to have 100% ROS production. Hydrogen peroxide (H₂O₂) was included as a positive control in preliminary experiments to verify assay responsiveness, and produced the expected concentration‐dependent increase in ROS production and decrease in cell viability. Results were expressed as the mean percentage of ROS production versus control group.

2.7. Determination of Antioxidant Enzyme Activities

Commercial kits were used to detect GPx1, GR, CAT, SOD, and TrxR activities.

GPx1 activity assay is performed based on the rapid and continuous reduction of oxidized glutathione (GSSG) generated during the GPx1 reaction by an excess of GR activity, which maintains a steady level of reduced glutathione [25]. T‐butyl hydroperoxide was utilized as a substrate, and spectrophotometric monitoring of the concurrent oxidation of NADPH was detected at 340 nm. One unit of enzyme was defined as the amount of GPx1 that transformed 1 μmole of NADPH to NADP per min at 37°C. The samples' GPx1 activity was expressed as U/mg protein.

CAT activity was determined with a stop solution to stop the enzyme‐mediated degradation of H₂O₂, and a chromogen solution (150 mM potassium phosphate buffer, pH 7.0, containing 2 mM 3,5‐dichloro‐2‐hydroxybenzenesulfonic acid and 0.25 mM 4‐aminoantipyrine) to detect the remaining H₂O₂ at 520 nm [26]. One unit of catalase was defined as the enzyme that decomposed one mM of H₂O₂ to oxygen and water per min at pH 7.0 at 25°C. The CAT activity was reported as nmol/min/mg protein.

The cell proliferation reagent (WST‐1), which is produced when a superoxide anion is reduced, was used to measure the SOD activity by a commercial kit. SOD decreased the rate of reduction with O₂, which was linearly correlated with the activity of xanthine oxidase (XO) [27]. Using this colorimetric technique, the IC₅₀ (50% inhibitory activity of SOD) was detected. The reduction in color development at 440 nm was used to measure the inhibition of SOD activity as an inhibitory activity because the absorbance at this wavelength was proportionate to the amount of superoxide anion. The SOD activity was expressed as U/mg protein.

The Thioredoxin Reductase Assay kit was used to colorimetrically measure the activity of TrxR. The process involved reducing 5,5′‐dithiobis‐(2‐nitrobenzoic) acid (DTNB) with NADPH to 5‐thio‐2‐nitrobenzoic acid (TNB), whose concentration was detected at 412 nm [28]. One unit of TrxR activity was defined as the amount of enzyme that caused an increase in absorbance of 1.0 per min and per ml at pH 7.0 at 25°C. The TrxR activity was expressed as U/mg protein.

GR activity was determined according to the reduction of GSSG to reduced glutathione [29]. Samples were added to a solution containing 1 mM GSSG, 100 mM phosphate buffer, and pH 7.5. 0.1 mM NADPH was added to initiate the reaction. For 5 min, the rate of NADPH oxidation was monitored at 340 nm. In order to account for nonspecific NADPH oxidation, controls were implemented. The amount of enzyme required to decrease 1 mM GSSG per min, which caused 1 mM NADPH to oxidize, was measured as one milligram of GR. GR activity was quantified as mU/mg protein.

2.8. Determination of Oxidative Stress Parameters

Glutathione, lipid peroxidation, and protein oxidation levels were determined by using commercial kits.

GSH levels were detected by the GSH Assay Kit. It is a kinetic assay in which the catalytic amounts of GSH caused a constant decrease of 5,5′‐dithiobis‐(2‐nitrobenzoic) acid (DTNB) to TNB at 412 nm [30]. Parallel measurements of a standard curve with known GSH values were used to achieve quantification, and the results were represented in nmol/mg protein.

The TBARS assay kit was utilized to quantify the levels of lipid peroxidation. This kit uses a spectrophotometric assay to determine the concentration of malondialdehyde (MDA), which is a key marker of lipid peroxidation. Under high temperature (90°C–100°C) and acidic conditions, MDA and thiobarbituric acid (TBA) form a complex, and the color intensity of the MDA‐TBA complex is determined spectrophotometrically at 540 nm [31]. A series of malondialdehyde (MDA) standards (0, 0.5, 5, 10, 20, 30, and 50 µM) was used to generate a standard curve, and sample MDA concentrations were calculated from this curve and expressed as µmol/L.

Protein carbonyl (PCO) content was measured using a “Protein Carbonyl Assay Kit” (Cayman Chemical, Ann Arbor, MI, USA), which is based on derivatization of protein carbonyl groups with 2,4‐dinitrophenylhydrazine (DNPH) to form stable dinitrophenyl (DNP) hydrazone adducts detected at 360 nm [32]. Results were expressed as nmol/mg protein.

Total antioxidant capacity (TAOC) represents the combined contribution of endogenous and dietary antioxidants. The assay is based on the oxidation of 2,2′‐azino‐di(3‐ethylbenzthiazoline sulfonate) (ABTS) mediated by metmyoglobin. Cellular antioxidants inhibit this oxidation reaction, thereby reflecting the antioxidant capacity of the sample [33]. By using Trolox, TAOC levels were determined at 750 nm and expressed as nmol/mg protein.

Total thiol (‐SH) levels were quantified using a Thiol Detection Assay Kit, which is based on the reaction of free thiol groups with Ellman's reagent (DTNB) to form 5‐thio−2‐nitrobenzoic acid (TNB), measured at 412 nm. Thiol content was calculated against cysteine standards and normalized to protein (nmol/mg protein).

2.9. Total Protein Determination

Protein content was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. In brief, bovine serum albumin (BSA) standards (0–2,000 µg/mL) were prepared, and a working reagent was obtained by mixing Reagent A and B at 50:1 (v/v). Samples and standards were incubated with the working reagent (typically 30 min at 37°C), and absorbance was read at 562 nm on a microplate reader; protein concentrations were calculated from the standard curve and used to normalize assay results (e.g., U/mg protein). The method relies on the reduction of Cu²⁺ to Cu⁺ by peptide bonds in alkaline medium (biuret reaction) and subsequent complexation of Cu⁺ by bicinchoninic acid, yielding a stable chromophore proportional to total protein.

2.10. Determination of DNA Damage

2.10.1. DNA Base Damage

After the specified incubation periods and trypsinization, cells were harvested, and total DNA was isolated using the Quick‐DNA Miniprep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. The procedure employs a genomic lysis buffer and spin‐column purification; DNA was eluted in nuclease‐free water. Levels of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a marker of oxidative DNA base damage, were then measured with a commercial sandwich ELISA kit (OXISResearch, OXIS Health Products Inc., Portland, OR, USA). Absorbance was read at 450 nm on a microplate reader (SpectraMax M2, Molecular Devices, San Jose, CA, USA), and concentrations were calculated from a four‐parameter logistic (4PL) standard curve generated in SoftMax Pro. Results were obtained as ng/mL of sample and, for comparability across assays, additionally normalized to total protein (Pierce BCA; reported as ng/mg protein).

2.10.2. Comet Assay

The comet assay (single‐cell gel electrophoresis) is a sensitive, rapid, and versatile method to quantify DNA strand breaks and alkali‐labile sites at the single‐cell level across diverse cell types; with enzyme‐modified versions, certain base lesions can also be detected. It is widely used both to evaluate DNA damage induced by environmental agents and to assess the protective effects of antioxidants/micronutrients [34, 35]. H₂O₂‐treated cells were used as a positive control and showed marked increases in % tail DNA, olive tail moment, tail length, and comet length under both alkaline and neutral conditions. The microfabricated CometChip adapts the assay to a 96‐well microwell array, improving throughput and reproducibility and enabling parallel measurements of DNA damage and repair [36, 37].

2.10.3. Alkaline Comet Assay

Single‐strand breaks (SSBs) were assessed using the alkaline CometChip assay, whereas double‐strand breaks (DSBs) were evaluated via the neutral CometChip protocol. For SSB analysis, treated HepG2 cells (100 µL, 10⁶ cells/mL) were dispensed into a bottomless 96‐well plate and allowed to settle by gravity. Following removal of the plate, the gel was overlaid with 1% low‐melting‐point agarose. Cells were subsequently loaded onto CometChips and subjected to lysis in a buffer containing 0.3 M NaOH, 2.5 M NaCl, 10 mM Tris, 1% Triton X‐100, and 1 mM Na₂EDTA. After overnight lysis, they were electrophoresed in a cold alkaline electrophoresis buffer (0.3 mol/L NaOH and 1 m mol/L Na₂EDTA) at 4°C for 40 min. The chips were then neutralized twice in fresh buffer (0.4 mol/L Tris‐HCl at pH 7.5) at 4°C for 15 min and stained with SYBR Gold according to the manufacturer's instructions for fluorescence imaging. Images were captured using a Nikon 80i upright microscope (Nikon Instruments, Tokyo, Japan) coupled with an automatic scanning system and analyzed with a customized Guicometanalyzer software by MATLAB (The Mathworks, Natick, MA).

2.10.4. Neutral Comet Assay

Double‐strand breaks (DSBs) were analyzed using the neutral CometChip assay. Gels were lysed in a neutral lysis buffer [2.5 M NaCl, 2 mM Na₂EDTA, 10 mM Tris, 1% N‐lauroylsarcosine, pH 9.5 with 0.5% Triton X‐100, and 10% DMSO added 20 min prior to use] at 43°C for 4 h. Following lysis, chips were washed three times with electrophoresis buffer (90 mM Tris, 90 mM boric acid, 2 mM Na₂EDTA, pH 8.5) for 30 min and electrophoresed at 4°C for 1 h. After neutralization, the CometChips were stained with SYBR Gold for fluorescence imaging. Image acquisition and analysis were performed in the same manner as described for the alkaline CometChip assay. DNA damage was quantified as % tail DNA and olive tail moment (OTM), representing the product of comet length and tail intensity.

2.11. Caspase Activities

Caspase‐3 activity was determined using a colorimetric caspase‐3 assay kit. The assay is based on the cleavage of the peptide substrate acetyl‐Asp‐Glu‐Val‐Asp p‐nitroanilide (Ac‐DEVD‐pNA) by caspase‐3, resulting in the release of the p‐nitroaniline (pNA) moiety. The concentration of released pNA was quantified either from absorbance measurements at 405 nm or via a calibration curve constructed with defined pNA standards. Similarly, caspase‐8 activity was measured using a colorimetric caspase‐8 assay kit. This assay relies on the hydrolysis of acetyl‐Ile‐Glu‐Thr‐Asp p‐nitroanilide (Ac‐IETD‐pNA) by caspase‐8, producing pNA, which was quantified based on absorbance at 405 nm or using a standard calibration curve. Staurosporine (1 µM) was used as a positive control and strongly increased caspase−3 activity, with a smaller but detectable effect on caspase−8.

2.12. Statistical Analysis

For statistical analysis, SPSS version 17.0 (SPSS Inc., Chicago, IL) was used. Comparisons among groups were performed using one‐way analysis of variance (ANOVA). For pairwise comparisons, Student's t‐test was used. The results were expressed as mean ± standard deviation (± SD). All graphical representations of experimental data were created using BioRender: Scientific Image and Illustration Software (Science Suite Inc., Toronto, Canada).

3. Results

3.1. Cell Viability

Aroclor 1254 reduced HepG2 cell viability in a concentration‐dependent manner (Figure 1A), and a concentration corresponding to approximately 70% viability was selected for subsequent mechanistic experiments. Curcumin alone maintained full viability at 5 µM, which was therefore chosen as the cytoprotective dose for all further assays (Figure 1B). H₂O₂ was used as a positive control and produced a concentration‐dependent reduction in cell viability ranging from mild to near‐complete cytotoxicity.

Figure 1.

Effects of Aroclor 1254 and curcumin on cell viability in HepG2 cells. (A) Effects of Aroclor 1254 on cell viability. (B) Effects of curcumin on cell viability. Values are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. Determination of Antioxidant Enzyme Activities

Exposure to Aroclor 1254 significantly suppressed all measured antioxidant enzyme activities, including GPx, GR, SOD, CAT and TrxR, compared to control (Figure 2A–E). Curcumin alone tended to enhance these activities above control levels. Co‐treatment with curcumin substantially restored enzyme activities toward or above control values, indicating a protective effect against Aroclor 1254‐induced impairment of antioxidant defenses. In H₂O₂‐treated cells, all antioxidant enzyme activities were markedly elevated compared to control, reflecting a compensatory activation of the enzymatic antioxidant defense system in response to oxidative challenge.

Figure 2.

Effects of curcumin and Aroclor 1254 on antioxidant enzyme activities in HepG2 cells. (A) Glutathione peroxidase (GPx, U/mg protein); (B) Glutathione reductase (GR, mU/mg protein); (C) Catalase (CAT, nmol/min/mg protein); (D) Superoxide dismutase (SOD, U/mg protein); (E) Thioredoxin reductase (TrxR, U/mg protein). Values are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.3. Determination of Oxidative Stress Parameters

Aroclor 1254 significantly altered the intracellular redox environment, increasing ROS, MDA/TBARS and protein carbonyl levels while depleting GSH, total thiols and TAOC compared to control (Figure 3A–F). Curcumin co‐treatment markedly attenuated these changes, reducing oxidative stress markers and partially restoring antioxidant capacity, although values did not fully return to control levels for all parameters. Curcumin alone had minimal effect on oxidative stress markers relative to control. In H₂O₂‐treated cells, oxidative damage was pronounced: lipid peroxidation and protein carbonyl levels were strongly elevated, while GSH, total thiols, and TAOC were markedly depleted, confirming the validity of H₂O₂ as a positive control for oxidative stress induction.

Figure 3.

Effects of curcumin and Aroclor 1254 on oxidative stress parameters in HepG2 cells. (A) Intracellular ROS (% of control); (B) Total glutathione, GSH (nmol/mg protein); (C) Malondialdehyde, MDA (µmol/L); (D) Protein carbonyls (nmol/mg protein); (E) Total antioxidant capacity, TAOC (nmol Trolox equivalents/mg protein); (F) Total thiol groups (nmol/mg protein). Values are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.4. DNA Damage

Aroclor 1254 significantly increased oxidative DNA damage, as evidenced by elevated 8‐OHdG levels and markedly altered comet assay parameters in both alkaline and neutral conditions compared to control (Figure 4A, Tables 1, 2). OGG1 levels did not differ significantly among the main treatment groups; however, H₂O₂‐treated cells showed a marked increase in OGG1 expression compared to control, curcumin, and curcumin and Aroclor 1254 groups (p < 0.01–0.001), indicating activation of base‐excision repair under H₂O₂‐induced oxidative stress (Figure 4B). Curcumin co‐treatment substantially attenuated Aroclor 1254‐induced DNA damage across all parameters, though values remained above control levels. Curcumin alone did not significantly affect DNA integrity relative to the control. In H₂O₂‐treated cells, 8‐OHdG levels and OGG1 expression were markedly elevated, and all comet assay parameters were strongly increased in both alkaline and neutral conditions, confirming pronounced induction of oxidative DNA damage and activation of base‐excision repair.

Figure 4.

Effects of curcumin and Aroclor 1254 on DNA damage markers. (A) 8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG) levels and (B) 8‐oxoguanine DNA glycosylase (OGG1) levels. Values are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Table 1.

Effects of curcumin and Aroclor 1254 on DNA damage parameters measured by alkaline comet assay.

	%tail DNA	Olive tail moment (OTM)	Tail length	Comet length
Control (C)	5.01 ± 0.21	10.31 ± 2.08	18.12 ± 2.4	39.12 ± 4.28
Curcumin group	4.08 ± 0.45*	8.44 ± 1.19	16.14 ± 2.28	28.14 ± 3.25*
Aroclor 1254 (A1254) group	12.25 ± 1.47***	30.18 ± 4.57***	32.21 ± 3.54***	56.39 ± 6.29***
Aroclor 1254 (A1254) + curcumin group	7.19 ± 0.67**	18.21 ± 3.18**	21.22 ± 3.13***	40.06 ± 4.23
H2O2	20.62 ± 2.88*	45.18 ± 4.47**	39.76 ± 2.45***	71.18 ± 8.84*

Note: Values are presented as mean ± SD (n = 3).

^* p < 0.05

^** p < 0.01

^*** p < 0.001.

Table 2.

Effects of curcumin and Aroclor 1254 on DNA damage parameters measured by neutral comet assay.

	%tail DNA	Olive tail moment (OTM)	Tail length	Comet length
Control (C)	10.21 ± 0.39	5.44 ± 0.49	13.06 ± 1.99	22.54 ± 3.82
Curcumin group	11.17 ± 1.27	6.18 ± 0.81	14.41 ± 3.56	25.18 ± 4.56*
Aroclor 1254 (A1254) group	37.39 ± 5.89*	39.56 ± 6.42*	45.18 ± 6.09*	59.23 ± 7.21**
Aroclor 1254 (A1254) + curcumin group	25.14 ± 5.43**	24.54 ± 4.52**	28.45 ± 4.96**	36.18 ± 5.19
H2O2	22.58 ± 3.76*	51.14 ± 5.99**	59.38 ± 7.18**	78.81 ± 10.74**

Note: Values are presented as mean ± SD (n = 3).

^* p < 0.05

^** p < 0.01.

3.5. Apoptotic Markers

Aroclor 1254 significantly increased both caspase‐3 and caspase‐8 activities compared to control and curcumin groups, indicating robust activation of both executioner and initiator apoptotic pathways (Figures 5A,B). Curcumin co‐treatment substantially reduced caspase activities toward control levels, suggesting a protective effect against Aroclor 1254‐induced apoptotic signaling. Curcumin alone did not significantly alter caspase activities relative to control. Staurosporine, used as a positive control, produced caspase‐3 activity below control levels while markedly elevating caspase‐8 activity, reflecting its known mechanism of preferential initiation of the extrinsic apoptotic pathway at the concentration used.

Figure 5.

Effects of curcumin and Aroclor 1254 on apoptotic markers. (A) Caspase‐3 activity and (B) Caspase‐8 activity. Values are presented as mean ± SD (n = 3). ***p < 0.001, ****p < 0.0001.

3.6. Mutagenicity Assessment

3.6.1. Ames Test

Aroclor 1254 induced concentration‐dependent increases in revertant colony numbers in both TA98 and TA100 strains of Salmonella typhimurium, under conditions with and without S9 metabolic activation (Table 3). Mutagenic potency was substantially enhanced in the presence of S9, suggesting that metabolic activation plays an important role in the genotoxic activity of Aroclor 1254. Both frameshift and base‐pair substitution mutation pathways were implicated, as indicated by the responses in TA98 and TA100 strains, respectively.

Table 3.

Mutagenicity assessment of Aroclor 1254 by Ames test.

Dose (µM)	A	B	C	D
Control	1.21 ± 0.18	1.28 ± 0.12	1.14 ± 0.23	1.21 ± 0.10
2.5	2.21 ± 1.15	3.42 ± 1.19	2.21 ± 1.15	2.21 ± 1.15
5	3.12 ± 0.88	3.56 ± 1.41	2.42 ± 0.46	3.02 ± 1.04
7.5	3.81 ± 0.45	4.77 ± 2.02	2.88 ± 0.54	4.15 ± 2.03
10	4.16 ± 1.02	5.01 ± 1.27	4.21 ± 0.69	5.12 ± 3.16
12.5	6.27 ± 1.19	9.91 ± 2.34	12.18 ± 2.45	13.01 ± 2.45
25	7.44 ± 2.01	12.24 ± 3.18	14.14 ± 2.61	13.99 ± 1.52

Note: Number of revertant colonies after application of different concentrations of Aroclor 1254 to Salmonella typhimurium strains: (A) TA98 without S9, (B) TA98 with S9, (C) TA100 without S9, and (D) TA100 with S9. Values are presented as mean ± SD (n = 3).

3.6.2. HPRT Mutation Assay

Aroclor 1254 exhibited concentration‐dependent cytotoxicity and mutagenicity in the HPRT assay (Table 4). Relative cell survival and cloning efficiency declined progressively with increasing concentrations, while mutation frequency increased substantially. The positive control MNNG produced more pronounced cytotoxic and mutagenic effects than Aroclor 1254, confirming the validity of the assay.

Table 4.

Mutagenicity assessment of Aroclor 1254 by HPRT gene mutation assay.

A1254 (µg/ml)	RCS (%)	CE (%)	NMC	MF (%) (/106)
0	100	95.56	4	6.21*
2.5	86.12	93.27	4	6.78*
5	80.21	92.18	5	7.42*
7.5	75.14	90.05	6	9.14*
10	64.17	89.32	8	19.21*
12.5	41.65	85.14	16	38.14*
25	35.18	80.14	24	41.67*
MNNG
0	100	11.27	6	45.21*
0.5	79.14	56.18	34	58.17*
1	29.41	40.21	38	77.15*
1.5	16.08	31.14	52	156.47*
2	5.01	18.75	103	543.01*

Note: Effects of various concentrations of Aroclor 1254 and MNNG (positive control) on relative cell survival (RCS, %), cloning efficiency (CE, %), number of mutant colonies (NMC), and mutation frequency (MF, per 10^6 cells). Values are presented as mean ± SD.

^*Significantly different from control (p < 0.05) (n = 3).

4. Discussion

In recent years, hepatotoxicity has been found to be mainly caused by apoptosis, oxidative stress, inflammation, and genotoxicity (14). The hepatoprotective effects of curcumin are largely attributed to its strong antioxidant properties. Notably, the methylene hydrogen and o‐methoxy phenolic groups constitute the primary structural components responsible for its free‐radical–scavenging activity. Curcumin exerts hepatoprotective and antioxidant effects through multiple complementary mechanisms, including direct neutralization of ROS, enhancement of endogenous antioxidant enzyme systems, modulation of key intracellular signaling pathways, and chelation of redox‐active metal ions. Together, these actions protect hepatocytes from oxidative stress–induced damage and subsequent cellular dysfunction [38].

This study provides comprehensive evidence that Aroclor 1254 induces profound biochemical and molecular perturbations characterized by oxidative imbalance, genotoxic insult, apoptotic activation, and mutagenicity, while curcumin exhibits a strong cytoprotective profile by counteracting these deleterious effects through multifactorial mechanisms. The significant downregulation of enzymatic antioxidants—namely SOD, CAT, GR, GPx, and TrxR—along with a marked decrease in TAOC levels, implicates that Aroclor 1254 can lead to the disruption of the endogenous oxidative defense network, potentially via ROS overproduction and redox‐sensitive signaling pathway dysregulation. In contrast, curcumin restored—and in some instances increased—antioxidant enzyme activities, consistent with a redox‐modulating effect. In parallel, oxidative injury indices increased (MDA, protein carbonyls, intracellular ROS), whereas endogenous antioxidant reserves declined (GSH, total thiols), and TAOC fell, collectively indicating substantial lipid peroxidation and protein oxidation. Curcumin's ability to reverse these alterations underscores its efficacy in preserving thiol redox status and maintaining cellular redox homeostasis. In the present study, we focused on a single curcumin concentration (5 µM) that did not affect cell viability and allowed detailed analysis of multiple oxidative, genotoxic, and apoptotic endpoints; future work may extend this approach to multiple curcumin doses in combination with Aroclor 1254 to define the protective dose–response relationship more comprehensively.

Several studies have demonstrated the cytotoxic and oxidative stress‐inducing effects of Aroclor 1254 across various cell types and organ systems. Salvi and Toninello (2001) demonstrated that Aroclor 1254 inhibits the mitochondrial permeability transition (MPT) in a dose‐dependent manner in isolated rat liver mitochondria, within a concentration range of 1 to 25 nmol/mg protein [39]. MPT is a well‐established event associated with necrotic and apoptotic cell death, often triggered by oxidative stress, ischemia‐reperfusion injury, and other cellular insults. Yuan et al. (2009) investigated the effects of Aroclor 1254 on HepG2 cells and observed a dose‐dependent increase in 7‐ethoxyresorufin‐O‐deethylase (EROD) activity, olive tail moment (a marker of DNA damage), and cytochrome P450 1A (CYP1A) enzymatic activity [40]. At higher concentrations (46 µM), Aroclor 1254 also significantly elevated 8‐OHdG levels, reflecting oxidative DNA damage. Similarly, Ghosh et al. (2010) showed that exposure of HepG2 and human kidney cells to PCB‐153 and PCB‐77 for varying durations (30 min to 48 h) induced pronounced cytotoxic and oxidative responses, highlighting the broader toxicological relevance of PCB congeners across diverse cell models [41]. Their results indicated that both PCB congeners induced time‐dependent apoptotic responses, underscoring the cytotoxic potential of PCBs through apoptotic pathways. In another study, Aly and Domènech (2009) treated isolated rat hepatocytes with Aroclor 1254 at concentrations ranging from 20 to 60 µM for 24 h [42]. Aroclor 1254 exposure resulted in a significant reduction in cell viability and increased lactate dehydrogenase (LDH) release into the culture medium in a dose‐dependent manner. Additionally, Aroclor 1254 significantly enhanced lipid peroxidation and increased the activities of EROD and pentoxyresorufin‐O‐deethylase (PROD), while markedly reducing GR, GP, and mitochondrial GSH levels. These findings indicate that mitochondria are primary targets of Aroclor 1254‐induced oxidative stress. Further extending the evidence of Aroclor 1254 toxicity beyond hepatic and renal systems, An et al. (2012) investigated its effects on MC3T3‐E1 preosteoblasts and demonstrated that concentrations above 15 mg/L significantly inhibited cell proliferation and viability, while inducing apoptosis in a time‐dependent manner. These cellular responses were accompanied by elevated ROS levels, reinforcing the role of oxidative stress as a central mechanism driving Aroclor 1254‐induced cytotoxicity in bone‐derived cells [7].

Sekaran et al. (2012) investigated the hepatotoxic potential of Aroclor 1254 in male Wistar rats and demonstrated that exposure resulted in pronounced hepatic oxidative injury, characterized by increased lipid peroxidation and depletion of endogenous antioxidant defenses [43]. Consistent with these findings, Buha et al. (2015) administered Aroclor 1254 orally to Wistar rats for 28 days and reported a clear dose‐dependent elevation in hepatic oxidative stress markers following subacute exposure [44]. Further extending the toxicological profile of Aroclor 1254 to developmental systems, Tang et al. (2015) exposed pregnant rats to Aroclor 1254 (1.5 mg/kg) from gestational day (GD) 6 through postnatal day (PND) 21 and observed significant biochemical and molecular disturbances indicative of developmental toxicity [45]. Their findings demonstrated that perinatal exposure to Aroclor 1254 induced pronounced oxidative and nitrosative stress in the brains of offspring by PND 22, largely due to decreased activities of critical antioxidant enzymes such as SOD and GPx. These observations are in line with our results and further strengthen the notion that PCB exposure, including Aroclor 1254, disrupts redox homeostasis and promotes oxidative damage across multiple organ systems. Supporting this evidence, Aydın et al. (2018) evaluated the genotoxic potential of Aroclor 1254 in various tissues of Sprague–Dawley rats and reported significant DNA damage, highlighting the broader systemic genotoxicity associated with PCB exposure. The animals were administered Aroclor 1254 at a dose of 10 mg/kg by oral gavage for 15 consecutive days. The results demonstrated that Aroclor 1254 exposure induced significant histopathological alterations in liver, kidney, and brain tissues. Aroclor 1254 treatment led to marked elevations in DNA damage parameters in liver tissues compared to the control group, with increases of 67%, 114%, and 384% in tail intensity, tail moment, and tail migration, respectively. These findings clearly indicate that Aroclor 1254 possesses strong genotoxic potential, affecting both somatic and germ cells [46]. Moreover, Balcı‐Özyurt et al. (2023) administered Aroclor 1254 (10 mg/kg/day) orally to rats for 15 days and reported pronounced alterations in liver histoarchitecture, elevated oxidative stress markers, and increased apoptosis [47]. In Aroclor 1254‐treated animals, relative liver weights and the activities of CAT, GPx1, and SOD were significantly higher than in controls, indicating a compensatory upregulation of antioxidant enzymes in response to heightened oxidative burden. Collectively, these findings, together with the broader body of experimental evidence, demonstrate that Aroclor 1254 exerts multi‐organ toxicity predominantly through oxidative stress mechanisms, affecting essential cellular targets such as mitochondria, DNA, and endogenous antioxidant defense systems.

Previous studies have employed a variety of animal models to elucidate the antioxidant activity of curcumin. Evidence from IUGR piglets, BALB/cJ mice, Wistar rats, and C57BL/6 mice consistently demonstrates that upregulation of the Nrf‐2 pathway plays a central role in mediating curcumin's antioxidant effects [48, 49, 50]. Beyond in vivo systems, several cell culture models have been used to further characterize its redox‐modulating properties. Lin and Chen (2008) reported that curcumin enhances intracellular GSH levels in a hepatic stellate cell (HSC) line, supporting its role in boosting non‐enzymatic antioxidant defenses [51]. Similarly, Shao et al. (2019) showed that curcumin upregulates the Nrf‐2 pathway in both HepG2 cells and human umbilical vein endothelial cells (HUVECs), reinforcing the notion that curcumin activates conserved cytoprotective signaling mechanisms across diverse cell types [19]. Taken together with our findings, these reports suggest that modulation of redox‐sensitive pathways—particularly Nrf2—may underlie the protective profile of curcumin, even though these upstream regulatory mechanisms were not directly assessed in our study.

Genotoxicity findings—reflected by increased 8‐OHdG levels and extensive DNA strand breaks detected in both alkaline and neutral comet assays—underscore the DNA‐damaging potential of Aroclor 1254, likely mediated through oxidative DNA adduct formation. Although OGG1 expression was not significantly altered, the marked reduction of DNA lesions following curcumin treatment suggests that its protective effects operate primarily through suppression of DNA damage rather than activation of base excision repair pathways. The observed upregulation of caspase‐3 and caspase‐8 activities after Aroclor 1254 exposure further confirms its pro‐apoptotic nature, indicating stimulation of both intrinsic and extrinsic apoptotic cascades; conversely, curcumin‐mediated inhibition of these enzymes highlights its anti‐apoptotic potential. Additionally, Aroclor 1254 displayed strong dose‐dependent mutagenic activity in both bacterial (Ames TA98/TA100) and mammalian (HPRT mutation) models, with effects amplified in the presence of S9 fraction, supporting the requirement for metabolic activation and formation of genotoxic intermediates. Although the direct antimutagenic activity of curcumin was not evaluated in this study, its substantial mitigation of oxidative and genotoxic stress suggests a likely indirect contribution to genomic stability.

In addition to ROS generation and lipid peroxidation, substantial genotoxic effects have been documented following PCB exposure. When PCBs undergo CYP‐mediated metabolic activation, they are converted into reactive arene oxide, semiquinone, or quinone intermediates. These electrophilic metabolites readily interact with nucleophilic sites in DNA—particularly guanine, the primary target—resulting in covalent adduct formation and subsequent genotoxicity. PCBs also undergo auto‐oxidation, producing ROS that further contribute to oxidative DNA damage [52]. Beyond point mutations, PCB‐induced genotoxicity manifests as atypical chromosomal segregation and the formation of DNA strand breaks. Previous studies have shown that Aroclor 1254, Aroclor 1221, and Aroclor 1260 induce genotoxic effects in Saccharomyces cerevisiae at high concentrations [53]. Aroclor 1254, in particular, has been demonstrated to cause DNA adduct formation, strand breakage, unscheduled DNA synthesis, and, to a lesser extent, chromosomal abnormalities in mammalian cell cultures in vitro. In vivo studies further support its mutagenic capacity: Aroclor 1254 increases chromosomal aberrations and micronucleus formation in fish, and elevates mutation frequencies in the livers of transgenic Big Blue® mice [54, 55, 56].

In a study evaluating the impact of prenatal PCB exposure and the potential protective effects of curcumin on DNA integrity, nine female and three male Wistar rats were allocated into three experimental groups and mated accordingly. From the second week of gestation to postnatal day 21, Group 1 received Aroclor 1254 (1 mg/kg), Group 2 received both Aroclor 1254 (1 mg/kg) and curcumin (250 mg/kg), and Group 3 received the vehicle (corn oil). Plasma 8‐OHdG concentrations were significantly higher in the Aroclor 1254 group compared with controls, indicating enhanced oxidative DNA damage. Histopathological alterations were also evident in the brain tissues of rat pups exposed to Aroclor 1254. Prenatal PCB exposure was shown to disrupt both DNA methylation and hydroxymethylation patterns and to induce DNA damage in the cerebral cortex and cerebellum. Notably, exogenous curcumin administration exerted a protective effect, mitigating PCB‐induced DNA damage in these brain regions [57].

5. Conclusion

This study demonstrates that Aroclor 1254 produced a broad toxicological signature with significant alterations in oxidative stress parameters and DNA damage, impairment of antioxidant defense systems, and promotion of genotoxic/mutagenic and apoptotic responses in HepG2 cells, Curcumin co‐treatment mitigated these effects at multiple levels by restoring antioxidant enzyme activities, lowering ROS, TBARS and carbonyls, replenishing total GSH and thiols, partially recovering TAOC, reducing 8‐OHdG and comet parameters, and blunting caspase activation, with several readouts returning toward control. Although protection was substantial, certain indices (e.g., TAOC, total thiols) did not fully normalize, indicating incomplete rescue under the tested conditions. Collectively, these findings designate Aroclor 1254 as a multifaceted hepatocellular toxicant and support curcumin as a promising protective adjunct that attenuates oxidative, genotoxic, and apoptotic outcomes in this model. Taken together, these findings highlight the need for careful safety evaluation of different PCB congeners in different in vitro and in vivo models and provide mechanistic insight that may guide future toxicological and regulatory assessments.

Author Contributions

Pınar Erkekoğlu: conceptualization, methodology, supervision, writing – review and editing, validation, resources. Selinay Başak Erdemli Köse: writing – original draft, investigation, methodology, formal analysis. Hülya Tezel Yalçın: writing – original draft, investigation. Deniz Arca Çakır: methodology, investigation, formal analysis, visualization, writing – original draft, writing – review and editing.

Funding

The authors have nothing to report.

Ethics Statement

This article does not contain any studies involving animals or human participants performed by any of the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.