Protective Effect of Thyme and Chestnut Honeys Enriched with Bee Products against Benzo(a)pyrene-Induced DNA Damage

The aim of the present study was to validate the cytotoxicity, genotoxicity, and preventive potential against benzo(a)pyrene (BaP)-induced DNA damage of nine samples of thyme and chestnut honeys enriched with bee products (royal jelly and propolis, 2–10%). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (0–250 mg/mL) to select nontoxic concentrations, and DNA damage (0.1–10 μg/mL) was evaluated by the alkaline single-cell gel electrophoresis or comet assay. Treatment with honey samples or royal jelly and propolis did not affect the viability of HepG2 cells up to 100 and 50 mg/mL, respectively. Treatment with 100 μM BaP significantly increased (p ≤ 0.001) the levels of the DNA strand breaks. None of the tested concentrations (0.1–10 μg/mL) of the honey samples (thyme and chestnut), royal jelly, and propolis caused DNA damage per se. All tested samples at all the concentrations used decreased the genotoxic effect of BaP. In addition, all mixtures of thyme or chestnut honeys with royal jelly or propolis showed a greater protective effect against BaP than the samples alone, being the thyme and chestnut honey samples enriched with 10% royal jelly and 10% propolis the most effective (70.4% and 69.4%, respectively). The observed protective effect may be associated with the phenolic content and antioxidant capacity of the studied samples. In conclusion, the thyme and chestnut honey samples enriched with bee products present potential as natural chemoprotective agents against the chemical carcinogen BaP.


Introduction
The European Union (EU) is the second honey producer worldwide after China, and Spain is the country with the largest honey production [1]. In addition to honey, Spain produces a variety of apiculture products, such as pollen, propolis, royal jelly, and beeswax [2]. Although the EU is an important honey producer (25,500 tons exported in 2021), EU member states imported 173,400 tons of honey in 2021, which came mostly from Ukraine (31%) [3]. In the context of the Ukraine war and climate change, sourcing significant volumes of honey required to replace the Ukrainian honey market is a great challenge [4]. Therefore, more efforts are needed to support local honey production. On the other hand, during the COVID-19 pandemic, the global honey market exhibited an increasing demand, and consumers are now asking for immunity-boosting and healthy food products [5,6]. In addition to the known effects of honey on the immune system, it has also shown antimicrobial, antioxidant, anticancer, antihyperlipidemic, and cardioprotective Thyme and chestnut honeys were mixed with royal jelly and propolis (2-10%). Samples (250 g) were homogenized in a laboratory blender, LB 400 W (with glass window in the door, blade speed (min −1 ): 240, fixed speed: 8 strokes/s). In order to optimize the extraction of antioxidant components and mixtures, propolis tincture was used and dissolved in 70% ecologic ethanol (La Alcoholera, La Rioja, Spain). To prepare honey, royal jelly, propolis, and mixture samples with 2 and 10% of royal jelly or propolis were weighed and diluted in sterile distilled water (1 g/mL). Then, sample solutions were sterilized by Millipore filtration (0.45 µm) and stored at −20 • C [25].
The 9 samples evaluated in the present work (Table 2) were previously selected from among 16 samples of honey mixtures with royal jelly and propolis (2-10%) for their high phenolic content and antioxidant capacity [25]. Table 2. Honey and bee product samples and mixtures selected from a previous work [25].

Code
Description Sample nº

HepG2 Cells
HepG2 cells were provided by Unidad de Terapias Farmacológicas and Unidad de Genética Molecular (Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Madrid, Spain). Only cells of passages 10-17 were used in these experiments. Cells were cultured as a monolayer in DMEM supplemented with 10% v/v heat-inactivated The alkaline comet assay is a technically simple, sensitive assay to detect DNA damage, such as strand breaks [28]. This section includes the Minimum Information for Reporting Comet Assay (MIRCA), a standardized reporting checklist for the description of comet assay procedures and results as a tool to ensure that the comet assay is performed rigorously and reported comprehensively [28]. The comet assay was carried out according to the protocol of Olive et al. (1992) [29]. Briefly, HepG2 cells were plated onto a 24-well plate at a density of 1.5 × 10 5 cells/mL in culture medium. Twenty-four hours after seeding, cells were exposed to thyme (TH) and chestnut (CH) honey samples and royal jelly (RJ) and propolis (PR) (0.1-10 µg/mL) for another 24 h at 37 • C and 5% CO 2 . Treatment with BaP (100 µM) was used as a positive control, and untreated cells (C 0 ) were used as a negative control. After incubation, 12 µL of a suspension of 1.5 × 10 5 cells was mixed with 70 µL of LMP agarose type VII (0.75% concentration in PBS) reaching a final concentration of agarose of 0.64%, distributed on slides (Diagnostic microscope slides, 3-well 14 mm, ER-203B-CE24, Thermo Scientific, Waltham, MA, USA) that had been precoated with LMP agarose type VII (0.30% concentration in PBS) and left to set on an ice tray. After solidification, cells were lysed in darkness for 1 h in a high salt alkaline buffer (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, 1% Triton X-100, pH 10). After incubation, slides were placed in electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH 13, cooled in a refrigerator) in darkness for 40 min. Electrophoresis was performed in a cold storage room, in darkness, in a Bio-Rad subcell GT unit containing the same buffer, for 30 min at 25 V. After electrophoresis, the slides were neutralized using 0.4 M Tris pH 7.5 and fixed in methanol. Subsequently, DNA was stained with GelRed ® 1X in Tris acetate EDTA (TAE 1×) for 5 min and examined in a fluorescence microscope (microscope magnification 10×) (OLYMPUS BH-2) connected to a computerized image analysis system (Komet 5.1). Results were expressed as percentage of Tail DNA.

Analysis of DNA Damage Induced by a Simultaneous Treatment with BaP and Honey Samples, Royal Jelly, Propolis, and Their Mixtures in the Alkaline Comet Assay
HepG2 cells were plated onto 24-well plates at a density of 1.5 × 10 5 cells/mL culture medium. Twenty-four hours after seeding, the samples ( Table 2) at 0.1-10 µg/mL were added to the wells, and plates were incubated for 24 h at 37 • C and 5% CO 2 . After incubation, cells were simultaneously treated with BaP (100 µM) and different concentrations of samples for another 24 h at 37 • C and 5% CO 2 . After the combined treatment of cells with BaP and samples, cells were processed as described above.

Statistical Analysis
Images of 50 randomly selected cells per concentration were evaluated, and the test was carried out three times. The reported Tail DNA is the mean ± standard error (SE) of three independent experiments. One-way analysis of variance (ANOVA) was carried out, and statistical comparisons of the different treatments were performed using Tukey's test. Values of p < 0.05 were considered statistically significant. All statistical analyses were performed using the Statgraphics Centurion 19 software (Statgraphics Technologies, Inc. The Plains, VA, USA). Figure 1 shows data on thyme (TH, sample 1) and chestnut (CH, sample 6) honey, royal jelly (RJ, sample 15), and propolis (PR, sample 16) cytotoxicity in HepG2 cells obtained by the MTT method. The studied sample concentrations were 0-250 mg/mL. The results show that as the concentration increases, the percentage of cell survival decreases. Concentrations of thyme and chestnut honey samples ( Figure 1A,B) up to 50 mg/mL did not affect the viability of HepG2 cells at 24, 48, and 72 h of treatment (>80%). When the cells were treated with monofloral honey samples at 100 mg/mL for 24 h, no significant differences were observed (p > 0.05) compared to the nontreated control cells (>75%). The treatment of cells with this concentration for longer periods of time caused a significant reduction in cell viability (p < 0.05). The results obtained in the present study are in agreement with those published by Al Refaey et al. (2021) but for Manuka honey [30]. These authors reported that concentrations up to 50 mg/mL of Manuka honey did not have a significant effect on the viability of HepG2 cells; however, higher concentrations of this type of honey incubated for 48 h had a significant reduction in cell viability [30]. In addition, a significant decrease in cell viability (p < 0.05) was observed at 250 mg/mL after treatment with the thyme (42%) and chestnut (30%) samples for 24 h. Recent studies have also evaluated the effect of different honey samples on HepG2 cell viability at similar concentrations and for the same periods of time [31,32]. Halawani (2021) observed a time-and dose-response effect when treating HepG2 cells at 24 h, 48 h, and 72 h with Manuka and Shaoka honeys from Saudi Arabia [32]. Manuka and Shaoka honey samples resulted more cytotoxic at lower concentrations compared to the thyme and chestnut honeys analyzed in the present study [32]. This may be due to the differences in the botanical and geographical origins of the bee product samples that have an influence on their composition and bioactive properties [33]. Royal jelly and propolis ( Figure 1C,D) up to a concentration of 100 mg/mL also did not reduce cell viability below 80% when cells were treated for 24 h. However, royal jelly showed a remarkable lower percentage of cell viability (5%) at the concentration of 250 mg/mL at all times tested (24, 48, and 72 h). Propolis was the only sample that at 250 mg/mL did not decrease cell viability (80%) when cells were treated for 24 and 48 h. In contrast, Badria et al. (2018) showed that lower concentrations (0.1 mg/mL) of Bulgarian, Egyptian, and Libyan propolis presented higher cytotoxic activity (7-85%) in HepG2 cells [34]. The authors suggest that the cytotoxic properties of the different propolis samples analyzed in their study can be correlated with their composition (specially flavonoids and total phenolic compounds) that depends on their geographical origin [33,34].

Evaluation of Cytotoxicity of Samples
On the other hand, the results of cytotoxicity of the monofloral honey samples enriched with royal jelly or propolis are shown in Figure 2. Concentrations up to 50 mg/mL of TH + 2PR, sample 4 ( Figure 2A), did not cause a significant effect (p > 0.05) on the cell viability of HepG2 cells. However, concentrations of 100 and 250 mg/mL at 48 and 72 h significantly decreased (p < 0.05) the viability to 20%. However, at 24 h, the viability of the cells was above 50%. When propolis was added to the thyme honey at 10% (TH + 10PR, sample 5, Figure 2B), all the concentrations tested at different incubation times did not reduce cell viability below 50%. However, when 10% propolis was added to the chestnut honey (CH + 10PR, sample 10, Figure 2C), concentrations over 50 mg/mL caused a significant decrease (p < 0.05) in HepG2 cell viability, reducing it below 40% when cells were treated for 48 and 72 h. When honey samples were enriched with both royal jelly and propolis ( Figure 2D,E), concentrations of 100 and 250 mg/mL for 24 h maintained cell viability over 50%, while reduced it to 25- 40% and 20-60% when cells were treated  during 48 or 72 h with TH + 10RJ + 10PR, sample 12 and CH + 10RJ + 10PR, sample 14,  respectively. To the best of our knowledge, there are no previously published studies  evaluating the effect of thyme and chestnut honeys enriched with royal jelly and propolis  on the viability of HepG2 cells. In line with previous results published for individual  monofloral honey samples, the concentrations of honeys enriched with royal jelly and propolis resulted in cytotoxicity to HepG2 cells when incubated at 50 mg/mL for more than 24 h [31]. Nguyen et al. (2021) showed that exposure of HepG2 cells to Manuka and other newly developed honeys (arjuna, guggul, jiaogulan, and olive) for 24 h, 48 h, and 72 h significantly reduced cell viability at 50 and 100 mg/mL [31]. Further research on the effect of monofloral honey enriched with other bee products, such as royal jelly and propolis, on the viability of cellular model is needed to compare the results obtained in the present study. On the other hand, the results of cytotoxicity of the monofloral honey samples enriched with royal jelly or propolis are shown in Figure 2. Concentrations up to 50 mg/mL of TH + 2PR, sample 4 ( Figure 2A), did not cause a significant effect (p > 0.05) on the cell viability of HepG2 cells. However, concentrations of 100 and 250 mg/mL at 48 and 72 h significantly decreased (p < 0.05) the viability to 20%. However, at 24 h, the viability of the cells was above 50%. When propolis was added to the thyme honey at 10% (TH + 10PR, sample 5, Figure 2B), all the concentrations tested at different incubation times did not reduce cell viability below 50%. However, when 10% propolis was added to the chestnut honey (CH + 10PR, sample 10, Figure 2C), concentrations over 50 mg/mL caused a significant decrease (p < 0.05) in HepG2 cell viability, reducing it below 40% when cells were treated for 48 and 72 h. When honey samples were enriched with both royal jelly and propolis ( Figure 2D,E), concentrations of 100 and 250 mg/mL for 24 h maintained cell viability over 50%, while reduced it to 25-40% and 20-60% when cells were treated during 48 or 72 h with TH + 10RJ + 10PR, sample 12 and CH + 10RJ + 10PR, sample 14, respectively. To the best of our knowledge, there are no previously published studies evaluating the effect of thyme and chestnut honeys enriched with royal jelly and propolis on the viability of HepG2 cells. In line with previous results published for individual monofloral honey samples, the concentrations of honeys enriched with royal jelly and propolis resulted in cytotoxicity to HepG2 cells when incubated at 50 mg/mL for more than 24 h [31]. Nguyen  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using Due to its wide range of applications in scientific research, the HepG2 cell line has gained popularity. Because this cell line has retained most of the metabolic functions of normal hepatocytes, it is used to study the toxic effects of different substances in vitro [35]. In this study, the cell viability assay performed in HepG2 cells was used to select nontoxic concentrations of samples to study their effect on DNA damage. In general, for all tested simples and enriched honey samples and for all incubation times, concentrations up to 10 mg/mL did not result in cytotoxicity to HepG2 cells. However, the tested honey samples, bee products, and their combinations at concentrations over 50 mg/mL affected the viability of HepG2 cells in a time-and dose-dependent manner (Figures 1 and 2). In agreement with our results, the final concentrations of honey samples used to study their effect on DNA damage were below 50 mg/mL, from 0.1 to 10 μg/mL [36].

Analysis of DNA Damage (DNA Strand Breaks) Induced by Thyme and Chestnut Honeys, Royal Jelly, and Propolis in the Alkaline Comet Assay
None of the honey samples (thyme and chestnut), royal jelly, and propolis at the tested concentrations (0.1-10 μg/mL) caused DNA damage per se ( Figure 3) because no significant differences were observed (p > 0.05) in the strand breaks when compared to the untreated control cells (C0). Thus, these concentrations were selected for subsequent studies, as these values are more likely to be achieved physiologically. As expected from our  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic ). C0, untreated cells. Asterisks indicate significant differences from the control. *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05.
Due to its wide range of applications in scientific research, the HepG2 cell line has gained popularity. Because this cell line has retained most of the metabolic functions of normal hepatocytes, it is used to study the toxic effects of different substances in vitro [35]. In this study, the cell viability assay performed in HepG2 cells was used to select nontoxic concentrations of samples to study their effect on DNA damage. In general, for all tested simples and enriched honey samples and for all incubation times, concentrations up to 10 mg/mL did not result in cytotoxicity to HepG2 cells. However, the tested honey samples, bee products, and their combinations at concentrations over 50 mg/mL affected the viability of HepG2 cells in a time-and dose-dependent manner (Figures 1 and 2). In agreement with our results, the final concentrations of honey samples used to study their effect on DNA damage were below 50 mg/mL, from 0.1 to 10 µg/mL [36]. Due to its wide range of applications in scientific research, the HepG2 cell line has gained popularity. Because this cell line has retained most of the metabolic functions of normal hepatocytes, it is used to study the toxic effects of different substances in vitro [35]. In this study, the cell viability assay performed in HepG2 cells was used to select nontoxic concentrations of samples to study their effect on DNA damage. In general, for all tested simples and enriched honey samples and for all incubation times, concentrations up to 10 mg/mL did not result in cytotoxicity to HepG2 cells. However, the tested honey samples, bee products, and their combinations at concentrations over 50 mg/mL affected the viability of HepG2 cells in a time-and dose-dependent manner (Figures 1 and 2). In agreement with our results, the final concentrations of honey samples used to study their effect on DNA damage were below 50 mg/mL, from 0.1 to 10 μg/mL [36].

Analysis of DNA Damage (DNA Strand Breaks) Induced by Thyme and Chestnut Honeys, Royal Jelly, and Propolis in the Alkaline Comet Assay
None of the honey samples (thyme and chestnut), royal jelly, and propolis at the tested concentrations (0.1-10 μg/mL) caused DNA damage per se ( Figure 3) because no significant differences were observed (p > 0.05) in the strand breaks when compared to the untreated control cells (C0). Thus, these concentrations were selected for subsequent studies, as these values are more likely to be achieved physiologically. As expected from our previous research [37,38], a significant increase (p < 0.05) in strand breaks was observed  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24]. Due to its wide range of applications in scientific research, the HepG2 cell line has gained popularity. Because this cell line has retained most of the metabolic functions of normal hepatocytes, it is used to study the toxic effects of different substances in vitro [35]. In this study, the cell viability assay performed in HepG2 cells was used to select nontoxic concentrations of samples to study their effect on DNA damage. In general, for all tested simples and enriched honey samples and for all incubation times, concentrations up to 10 mg/mL did not result in cytotoxicity to HepG2 cells. However, the tested honey samples, bee products, and their combinations at concentrations over 50 mg/mL affected the viability of HepG2 cells in a time-and dose-dependent manner (Figures 1 and 2). In agreement with our results, the final concentrations of honey samples used to study their effect on DNA damage were below 50 mg/mL, from 0.1 to 10 μg/mL [36].

Analysis of DNA Damage (DNA Strand Breaks) Induced by Thyme and Chestnut Honeys, Royal Jelly, and Propolis in the Alkaline Comet Assay
None of the honey samples (thyme and chestnut), royal jelly, and propolis at the tested concentrations (0.1-10 μg/mL) caused DNA damage per se ( Figure 3) because no significant differences were observed (p > 0.05) in the strand breaks when compared to the untreated control cells (C0). Thus, these concentrations were selected for subsequent studies, as these values are more likely to be achieved physiologically. As expected from our previous research [37,38], a significant increase (p < 0.05) in strand breaks was observed  Exposure to environmental toxicants induces oxidative stress, leading to DNA d age induced by ROS [39]. DNA damage can interfere with essential cellular proces lead to cell damage, and induce mutations that contribute to aging and diseases [40]. cording to the European Food Safety Authority (EFSA), genotoxicity studies are used risk assessments of substances present in food to identify compounds responsible for itable damage in humans, predict potential genotoxic, and contribute to the understa ing of the mechanisms of action of chemical carcinogens [41]. The comet assay is a ra sensitive, and relatively simple method recommended for detecting DNA damage at level of individual cells [42]. In this work, the comet assay was used to evaluate the g toxic effect of the individual samples ( Figure 3). None of them at the tested concentrat produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in total polyphenol content and the antioxidant capacity of different kinds of honey [25 To the best of our knowledge, no previous research has reported the effect of monofl thyme or chestnut honey samples on DNA damage by the comet assay. Other monofl (rosemary and heather) and heterofloral honey samples were also not genotoxic at centrations higher than those used in this study [44]. On the other hand, the genot effect of royal jelly and propolis has been previously studied. The genotoxic effect of r jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo u animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at ). C0, untreated cells. Asterisks indicate significant difference from control. *** p ≤ 0.001.

Analysis of DNA Damage (DNA Strand Breaks) Induced by Thyme and Chestnut Honeys, Royal Jelly, and Propolis in the Alkaline Comet Assay
None of the honey samples (thyme and chestnut), royal jelly, and propolis at the tested concentrations (0.1-10 µg/mL) caused DNA damage per se ( Figure 3) because no significant differences were observed (p > 0.05) in the strand breaks when compared to the untreated control cells (C0). Thus, these concentrations were selected for subsequent studies, as these values are more likely to be achieved physiologically. As expected from our previous research [37,38], a significant increase (p < 0.05) in strand breaks was observed when BaP 100 µM, used as the positive control (C1), was added to the HepG2 cells (40% tail DNA).
Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 μg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 μg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 μg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 μg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 μg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 μg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 μg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment ). Asterisks indicate significant difference from control (C 0 ). *** p ≤ 0.001.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using animal models. In rats, the administration of Egyptian royal jelly at 100 and 250 mg/kg body weight for 5 days did not induce DNA damage in liver cells [45]. In addition, the peripheral blood cells of mice treated for 24 and 48 h with commercial royal jelly at 150, 300, and 1000 mg/kg did not show significant differences in DNA damage when compared to the control with untreated mice [24].
In agreement with our results, a recent study has shown the nongenotoxic effect of propolis at 10 µg/mL after 24 h treatment of HepG2 cells [22]. In addition, the treatment of other hepatocytes (ZF-L) with higher concentrations of a Brazilian red propolis ethanolic extract (100-500 µg/mL) for 2 h showed no genotoxic effect [46]. In contrast, similar concentrations of propolis to those used in the present study have caused genotoxic effects on fibroblast synovial cells [47]. Gajek et al. (2020) indicated that the compound responsible for this genotoxic effect is caffeic acid phenethyl ester (CAPE), an active component of propolis, when tested on different gastrointestinal neoplastic cell lines [48]. These different effects observed for propolis are expected because the characteristics of this bee product are associated with its region of origin and botanical source that determine its chemical composition [12].

Analysis of DNA Damage (DNA Strand Breaks) Induced by a Simultaneous Treatment of BaP and Thyme and Chestnut Honey Samples and Honey Enriched with Royal Jelly and Propolis in the Alkaline Comet Assay
The effect of thyme and chestnut honeys, royal jelly, and propolis on BaP-induced DNA damage in HepG2 cells is shown in Figure 4. All samples tested at all concentrations used (0.1-10 µg/mL) decreased the genotoxic effect of BaP 100 µM. Among the studied samples, propolis showed the highest percentage of reduction (56.3-57.7%) at all tested concentrations ( Figure 4D). Our previous research indicated that propolis possesses the highest antioxidant properties compared to the other studied bee products [25], which may be the reason why propolis has shown the greatest potential for reducing induced DNA damage. In addition, royal jelly and chestnut and thyme honeys at 10 µg/mL significantly reduced DNA strand breaks (p < 0.05) induced by BaP in a 56.3%, 55%, and 49.7%, respectively. on fibroblast synovial cells [47]. Gajek et al. (2020) indicated that the compound responsible for this genotoxic effect is caffeic acid phenethyl ester (CAPE), an active component of propolis, when tested on different gastrointestinal neoplastic cell lines [48]. These different effects observed for propolis are expected because the characteristics of this bee product are associated with its region of origin and botanical source that determine its chemical composition [12].

Analysis of DNA Damage (DNA Strand Breaks) Induced by a Simultaneous Treatment of BaP and Thyme and Chestnut Honey Samples and Honey Enriched with Royal Jelly and Propolis in the Alkaline Comet Assay
The effect of thyme and chestnut honeys, royal jelly, and propolis on BaP-induced DNA damage in HepG2 cells is shown in Figure 4. All samples tested at all concentrations used (0.1-10 μg/mL) decreased the genotoxic effect of BaP 100 μM. Among the studied samples, propolis showed the highest percentage of reduction (56.3-57.7%) at all tested concentrations ( Figure 4D). Our previous research indicated that propolis possesses the highest antioxidant properties compared to the other studied bee products [25], which may be the reason why propolis has shown the greatest potential for reducing induced DNA damage. In addition, royal jelly and chestnut and thyme honeys at 10 μg/mL significantly reduced DNA strand breaks (p < 0.05) induced by BaP in a 56.3%, 55%, and 49.7%, respectively. The protective effect of honey samples enriched with royal jelly or propolis on BaPinduced DNA damage is shown in Figure 5. All mixtures of thyme or chestnut honey samples with royal jelly or propolis showed a greater protective effect against BaP than osure to environmental toxicants induces oxidative stress, leading to DNA damced by ROS [39]. DNA damage can interfere with essential cellular processes, ll damage, and induce mutations that contribute to aging and diseases [40]. Aco the European Food Safety Authority (EFSA), genotoxicity studies are used for sments of substances present in food to identify compounds responsible for hermage in humans, predict potential genotoxic, and contribute to the understandmechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, , and relatively simple method recommended for detecting DNA damage at the ndividual cells [42]. In this work, the comet assay was used to evaluate the genoct of the individual samples ( Figure 3). None of them at the tested concentrations DNA damage compared to the untreated control cells. botanical and the geographical origins of honey contribute to variations in the phenol content and the antioxidant capacity of different kinds of honey [25,43]. st of our knowledge, no previous research has reported the effect of monofloral chestnut honey samples on DNA damage by the comet assay. Other monofloral y and heather) and heterofloral honey samples were also not genotoxic at conns higher than those used in this study [44]. On the other hand, the genotoxic oyal jelly and propolis has been previously studied. The genotoxic effect of royal not been evaluated in vitro by the comet assay, but has been studied in vivo using  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic effect of royal jelly and propolis has been previously studied. The genotoxic effect of royal jelly has not been evaluated in vitro by the comet assay, but has been studied in vivo using  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. To the best of our knowledge, no previous research has reported the effect of monofloral thyme or chestnut honey samples on DNA damage by the comet assay. Other monofloral (rosemary and heather) and heterofloral honey samples were also not genotoxic at concentrations higher than those used in this study [44]. On the other hand, the genotoxic  Exposure to environmental toxicants induces oxidative stress, leading to DNA d age induced by ROS [39]. DNA damage can interfere with essential cellular proce lead to cell damage, and induce mutations that contribute to aging and diseases [40] cording to the European Food Safety Authority (EFSA), genotoxicity studies are use risk assessments of substances present in food to identify compounds responsible for itable damage in humans, predict potential genotoxic, and contribute to the underst ing of the mechanisms of action of chemical carcinogens [41]. The comet assay is a ra sensitive, and relatively simple method recommended for detecting DNA damage a level of individual cells [42]. In this work, the comet assay was used to evaluate the g toxic effect of the individual samples ( Figure 3). None of them at the tested concentra produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in total polyphenol content and the antioxidant capacity of different kinds of honey [25 To the best of our knowledge, no previous research has reported the effect of monof thyme or chestnut honey samples on DNA damage by the comet assay. Other monof (rosemary and heather) and heterofloral honey samples were also not genotoxic at centrations higher than those used in this study [44]. On the other hand, the geno  Exposure to environmental toxicants induces oxidative stress, leadin age induced by ROS [39]. DNA damage can interfere with essential cel lead to cell damage, and induce mutations that contribute to aging and di cording to the European Food Safety Authority (EFSA), genotoxicity stud risk assessments of substances present in food to identify compounds resp itable damage in humans, predict potential genotoxic, and contribute to t ing of the mechanisms of action of chemical carcinogens [41]. The comet a sensitive, and relatively simple method recommended for detecting DNA level of individual cells [42]. In this work, the comet assay was used to eva toxic effect of the individual samples ( Figure 3). None of them at the tested produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to v total polyphenol content and the antioxidant capacity of different kinds o To the best of our knowledge, no previous research has reported the effec thyme or chestnut honey samples on DNA damage by the comet assay. O (rosemary and heather) and heterofloral honey samples were also not ge centrations higher than those used in this study [44]. On the other hand ) Cells treated with BaP (100 µM) and RJ. ( onmental toxicants induces oxidative stress, leading to DNA dam-39]. DNA damage can interfere with essential cellular processes, d induce mutations that contribute to aging and diseases [40]. Acn Food Safety Authority (EFSA), genotoxicity studies are used for stances present in food to identify compounds responsible for herns, predict potential genotoxic, and contribute to the understandof action of chemical carcinogens [41]. The comet assay is a rapid, y simple method recommended for detecting DNA damage at the [42]. In this work, the comet assay was used to evaluate the genoidual samples (Figure 3). None of them at the tested concentrations e compared to the untreated control cells. the geographical origins of honey contribute to variations in the nt and the antioxidant capacity of different kinds of honey [25,43].
ledge, no previous research has reported the effect of monofloral ey samples on DNA damage by the comet assay. Other monofloral ) and heterofloral honey samples were also not genotoxic at con-) Cells treated with BaP (100 µM) and PR. Asterisks indicate significant difference from control (C 1 ). *** p ≤ 0.001 and ** p ≤ 0.01.
The protective effect of honey samples enriched with royal jelly or propolis on BaPinduced DNA damage is shown in Figure 5. All mixtures of thyme or chestnut honey samples with royal jelly or propolis showed a greater protective effect against BaP than the samples alone. The thyme and chestnut honey samples enriched with 10% royal jelly and 10% propolis ( Figure 5D,E) showed the highest protective effect against BaP (70.4% and 69.4%, respectively). Figure 6 shows the typical comet assay images after treatment with BaP (100 µM) and the absence of comets after exposure to BaP and samples 12 and 14. Thyme honey enriched with 2 or 10% propolis ( Figure 5A,B) had very similar values (69% and 67.8%) as did the chestnut honey sample enriched with 10% propolis (67.8%). This is the first time the chemoprotective effect of enriched honey mixtures has been evaluated on induced DNA damage. In this study, the observed reduction in induced DNA damage by BaP by both honey samples alone and mixed with other bee products was about two times higher than that reported for other monofloral (rosemary and heather) and heterofloral kinds of honey [44]. Different chemoprotective effects have been observed depending on the botanical origin of honey samples. Rosemary honey at 0.1-10 mg/mL decreased DNA strand breaks induced by BaP in a dose-dependent manner (19-25%), and heather honey prevented DNA strand breaks only at the lowest concentration (0.1 mg/mL, 15%) used in that study [44]. Heterofloral honey reached a maximum decrease in DNA strand breaks at the highest concentration used of 10 mg/mL (34%). Because an artificial honey sample did not show any protective effects against DNA damage induced by BaP, polyphenols might be, at least partially, the potential chemopreventive agents present in honey that protect against damage induced by BaP [44]. In fact, it has been described that due to their polyphenolic profile, monofloral honey samples have a significant antioxidant capacity, as well as antidiabetic, antimicrobial, and anticancer properties [49].
with BaP (100 μM) and the absence of comets after exposure to BaP and samples 12 and 14. Thyme honey enriched with 2 or 10% propolis ( Figure 5A,B) had very similar values (69% and 67.8%) as did the chestnut honey sample enriched with 10% propolis (67.8%). This is the first time the chemoprotective effect of enriched honey mixtures has been evaluated on induced DNA damage. In this study, the observed reduction in induced DNA damage by BaP by both honey samples alone and mixed with other bee products was about two times higher than that reported for other monofloral (rosemary and heather) and heterofloral kinds of honey [44]. Different chemoprotective effects have been observed depending on the botanical origin of honey samples. Rosemary honey at 0.1-10 mg/mL decreased DNA strand breaks induced by BaP in a dose-dependent manner (19-25%), and heather honey prevented DNA strand breaks only at the lowest concentration (0.1 mg/mL, 15%) used in that study [44]. Heterofloral honey reached a maximum decrease in DNA strand breaks at the highest concentration used of 10 mg/mL (34%). Because an artificial honey sample did not show any protective effects against DNA damage induced by BaP, polyphenols might be, at least partially, the potential chemopreventive agents present in honey that protect against damage induced by BaP [44]. In fact, it has been described that due to their polyphenolic profile, monofloral honey samples have a significant antioxidant capacity, as well as antidiabetic, antimicrobial, and anticancer properties [49].  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations produced DNA damage compared to the untreated control cells.
The botanical and the geographical origins of honey contribute to variations in the total polyphenol content and the antioxidant capacity of different kinds of honey [25,43]. s induces oxidative stress, leading to DNA damcan interfere with essential cellular processes, s that contribute to aging and diseases [40]. Acthority (EFSA), genotoxicity studies are used for food to identify compounds responsible for herial genotoxic, and contribute to the understandical carcinogens [41]. The comet assay is a rapid, ecommended for detecting DNA damage at the , the comet assay was used to evaluate the genoure 3). None of them at the tested concentrations untreated control cells. origins of honey contribute to variations in the nmental toxicants induces oxidative stress, leading to DNA dam-9]. DNA damage can interfere with essential cellular processes, induce mutations that contribute to aging and diseases [40]. Ac-Food Safety Authority (EFSA), genotoxicity studies are used for tances present in food to identify compounds responsible for hers, predict potential genotoxic, and contribute to the understandf action of chemical carcinogens [41]. The comet assay is a rapid, simple method recommended for detecting DNA damage at the [42]. In this work, the comet assay was used to evaluate the genoual samples (Figure 3). None of them at the tested concentrations  Exposure to environmental toxicants induces oxidative age induced by ROS [39]. DNA damage can interfere with lead to cell damage, and induce mutations that contribute t cording to the European Food Safety Authority (EFSA), gen risk assessments of substances present in food to identify co itable damage in humans, predict potential genotoxic, and ing of the mechanisms of action of chemical carcinogens [41 sensitive, and relatively simple method recommended for d level of individual cells [42]. In this work, the comet assay w toxic effect of the individual samples ( Figure 3). None of the produced DNA damage compared to the untreated control  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations  Exposure to environmental toxicants induces oxidative stress, leading to DNA damage induced by ROS [39]. DNA damage can interfere with essential cellular processes, lead to cell damage, and induce mutations that contribute to aging and diseases [40]. According to the European Food Safety Authority (EFSA), genotoxicity studies are used for risk assessments of substances present in food to identify compounds responsible for heritable damage in humans, predict potential genotoxic, and contribute to the understanding of the mechanisms of action of chemical carcinogens [41]. The comet assay is a rapid, sensitive, and relatively simple method recommended for detecting DNA damage at the level of individual cells [42]. In this work, the comet assay was used to evaluate the genotoxic effect of the individual samples ( Figure 3). None of them at the tested concentrations ) Cells treated with BaP (100 µM) and CH + 10RJ + 10PR. Asterisks indicate significant difference from control (C 1 ). *** p ≤ 0.001.
Even though honey is recognized as one of the first known functional foods and has been widely used in traditional medicine for centuries (apitherapy) [7,50], the scientific community is still interested in exploring further health-promoting properties honey may possess [50]. In order to clarify to honey consumers what honey's total potential is, a newly joined property named "Power of Honey" considering honey's antibacterial and antioxidant properties have been recently defined [50]. This novel approach successfully predicted the greater Power of Honey in forest honey compared to acacia, sunflower, and meadow honey samples [50]. The high antioxidant capacity of honey and the other bee products studied in the present work plays an important physiological role due to its capacity to scavenge hydroxyl and superoxide radicals related to cell and DNA damage and related diseases [12]. In this context, genotoxicity tests are also useful for assisting in the discovery of genoprotective substances for DNA mutation prevention [46]. In the present study, all tested individual samples at all concentrations used (0.1-10 µg/mL) decreased the genotoxic effect produced by BaP (Figure 4). In addition, the mixtures of thyme honey or chestnut honey with royal jelly or propolis showed a greater protective effect against BaP than the samples alone ( Figure 6).  Even though honey is recognized as one of the first known functional foods and has been widely used in traditional medicine for centuries (apitherapy) [7,50], the scientific community is still interested in exploring further health-promoting properties honey may possess [50]. In order to clarify to honey consumers what honey's total potential is, a newly joined property named "Power of Honey" considering honey's antibacterial and antioxidant properties have been recently defined [50]. This novel approach successfully predicted the greater Power of Honey in forest honey compared to acacia, sunflower, and meadow honey samples [50]. The high antioxidant capacity of honey and the other bee products studied in the present work plays an important physiological role due to its capacity to scavenge hydroxyl and superoxide radicals related to cell and DNA damage and related diseases [12]. In this context, genotoxicity tests are also useful for assisting in the discovery of genoprotective substances for DNA mutation prevention [46]. In the present study, all tested individual samples at all concentrations used (0.1-10 μg/mL) decreased the genotoxic effect produced by BaP (Figure 4). In addition, the mixtures of thyme honey or chestnut honey with royal jelly or propolis showed a greater protective effect against BaP than the samples alone ( Figure 6).
The observed protective effects may be associated with the phenolic content and antioxidant capacity of the studied samples that have been recently reported in our previous study [25]. The antioxidant potential of these honey samples has been confirmed by the analysis of their total phenolic content analyzed using the Folin-Ciocalteu reagent and antioxidant capacity by three different assays (DPPH, ABTS, and ORAC) to consider the complexity of the food matrix, the chemical diversity of antioxidants, and the different mechanisms of oxidative processes. A significant positive correlation was observed between total phenolic compounds and the antioxidant capacity analyzed by the three methods, suggesting that the antioxidant capacity could be attributed, at least partially, to the phenolic compounds present in the samples [25]. Our previous research indicated that mixtures of honey with 10% of propolis increased the value of total phenolic content and antioxidant capacity compared to natural honey and therefore were chosen to be analyzed in the present study. In addition, these samples have shown the potential to reduce ROS production in the same human hepatoma cells (HepG2 cell line) [25]. Other Spanish honey samples, such as heather, rosemary, and heterofloral, have also shown the ability to significantly decrease intracellular ROS levels in a time-dependent manner in HL-60 cells [51]. Chestnut honey enriched with 10% RJ and 10% PR (sample 14) was the most effective The observed protective effects may be associated with the phenolic content and antioxidant capacity of the studied samples that have been recently reported in our previous study [25]. The antioxidant potential of these honey samples has been confirmed by the analysis of their total phenolic content analyzed using the Folin-Ciocalteu reagent and antioxidant capacity by three different assays (DPPH, ABTS, and ORAC) to consider the complexity of the food matrix, the chemical diversity of antioxidants, and the different mechanisms of oxidative processes. A significant positive correlation was observed between total phenolic compounds and the antioxidant capacity analyzed by the three methods, suggesting that the antioxidant capacity could be attributed, at least partially, to the phenolic compounds present in the samples [25]. Our previous research indicated that mixtures of honey with 10% of propolis increased the value of total phenolic content and antioxidant capacity compared to natural honey and therefore were chosen to be analyzed in the present study. In addition, these samples have shown the potential to reduce ROS production in the same human hepatoma cells (HepG2 cell line) [25]. Other Spanish honey samples, such as heather, rosemary, and heterofloral, have also shown the ability to significantly decrease intracellular ROS levels in a time-dependent manner in HL-60 cells [51]. Chestnut honey enriched with 10% RJ and 10% PR (sample 14) was the most effective in the reduction in intracellular ROS, and it even improved the results obtained for N-acetyl-L-cysteine used as an antioxidant control [25]. In this study, sample 14, together with sample 12 (TH + 10RJ + 10PR), was also the most effective in preventing induced DNA damage ( Figure 6).
Honey is a good source of natural antioxidants whose activity is mainly due to the presence of phenolic compounds, although amino acids, proteins, vitamins, and carotenoid derivatives also contribute to the antioxidant capacity of this food product [25,43]. Polyphenols, and more specifically flavonols and flavanols, such as quercetin and myricetin, and catechin and epicatechin, respectively, protect human-derived cells against DNA strand breaks and oxidative DNA damage effects induced by BaP [38]. These compounds have been found both in thyme [52][53][54] and chestnut [49,55] honey samples, and therefore, the observed protective effect against induced DNA damage can be associated, at least par-tially, to these compounds. Polyphenols are the main constituents of propolis that have health-promoting properties, and propolis has been reported as the bee product containing the highest number of phenolic compounds [56]. Polyphenols, such as quercetin [57] and catechin [12], that have shown protective effects against this specific carcinogen, have been reported in this bee product. In addition, CAPE, known as a key anticancer component [58], has been ascribed as the most valuable biologically active compound in propolis for its several biological and pharmacological properties [56]. This compound has shown to effectively prevent cellular DNA damage induced by overloaded iron through decreasing the labile iron pool levels in HeLa cells [59]. Polyphenols are also present in royal jelly [25,60], but proteins are the most abundant components (50% dry weight), with major royal jelly proteins (MRJPs) being the most important (80-90%) [14]. These proteins also protect against DNA damage induced by ROS, acting as scavengers against superoxide anions and hydroxyl radicals [61].
According to Patra et al. (2021), dietary phytochemicals are "non-nutritive, relatively non-toxic, disease preventive phytoconstituents that are safe to consume", and including them in a healthy diet can prevent about 30% of carcinogenesis [62]. Dietary polyphenols have an important role in cancer prevention by their antiproliferative and apoptotic properties, among others [62]. In this context, three types of crude Spanish commercial honey samples (heather, rosemary, and heterofloral) induced apoptosis in a concentration-and time dependent-manner. Heather and heterofloral honey samples possessed the highest phenolic content and were the most effective to induce apoptosis in human peripheral blood promyelocytic leukemia cells (HL-60 cells) [51]. These same honey samples have shown protective effects against acrylamide-induced cytotoxicity [63]. Because an artificial honey sample did not mitigate the cytotoxic effect induced by acrylamide, the protective effect of these honeys may be attributed to their polyphenolic content and not to the sugar constituents present in this food matrix [63]. In addition to protecting against induced DNA damage, the chestnut honey studied in the present work may also present anticancer effects by promoting apoptosis due to the presence of 3-2 -pyrrilonidinyl-kynurenic acid (3-PKA-L), an alkaloid usually present in this monofloral honey, that has shown proapoptotic properties [64]. Further studies demonstrating the potential of monofloral honey samples as healthy dietary ingredients with chemopreventive properties are needed to support local honey production and respond to the increasing honey demand of consumers derived from the COVID-19 pandemic.

Conclusions
In this work, we proved the noncytotoxic and nongenotoxic characteristics of monofloral honey samples enriched with royal jelly and propolis in order to give more information related to the safety and chemoprotective potential of these enriched products. Our results indicate, for the first time, that thyme and chestnut honeys, royal jelly, propolis, and their combination protect human cells from DNA strand breaks induced by BaP. Thyme and chestnut honey samples enriched with 10% royal jelly and 10% propolis showed the highest protective effect against food mutagen-induced DNA damage in HepG2 cells. Polyphenols present in these samples and their antioxidant capacity are partially responsible for the observed chemoprotective effect.