AbstractUranium compounds, particularly uranyl acetate, are known to cause significant genotoxic and oxidative damage in biological systems due to their high chemical reactivity. In recent years, plant-based antioxidants, such as those found in Abelmoschus esculentus (L.) Moench, have attracted considerable attention for their potential to mitigate the toxicity of heavy metals. The present study was conducted to investigate the mitigative effect of A. esculentus (L.) Moench extract on the toxicity induced by uranyl acetate in the model organism Allium cepa L. Tap water, 250 mg L− 1A. esculentus extract, 500 mg L− 1A. esculentus extract, 0.1 mg mL− 1 uranyl acetate, 0.1 mg mL− 1 uranyl acetate + 250 mg L− 1A. esculentus extract, and 0.1 mg mL− 1 uranyl acetate + 500 mg L− 1A. esculentus extract were administered to the six groups of A. cepa bulbs. The group treated with tap water served as control group. Uranyl acetate caused a significant reduction in rooting percentage, root elongation, weight gain, mitotic index and the levels of chlorophyll a and chlorophyll b. There was a notable increase in the frequency of micronuclei and chromosomal aberrations (CAs), as well as a rise in malondialdehyde level following the uranyl acetate administration. The uranyl acetate-induced CAs included fragment, sticky chromosome, vagrant chromosome, bridge and unequal distribution of chromatin. The group treated with uranyl acetate also exhibited elevated levels of DNA damage, meristematic cell injury and superoxide dismutase and catalase enzyme activities. The meristematic damage induced by uranyl acetate was observed in the epidermis, cortex and nucleus of epidermal cells. The A. esculentus extract was observed to possess high levels of phenolic compounds and exhibited dose-dependent efficacy in mitigating the adverse effects of uranyl acetate. According to LC/MS analysis, the most abundant phenolic compounds in A. esculentus extract were rutin, caffeic acid, quercetin, salicylic acid and 4-OH benzoic acid. It was concluded that the capacity of A. esculentus extract to reduce uranyl acetate-induced multidirectional toxicity may be related to the ability of its phenolic compounds to chelate and scavenge radicals.IntroductionUranium (U) is defined as a radioactive heavy metal with a silvery-white color, which has existed on this planet since its formation. Due to its radioactive and chemical properties, the substance is used extensively in industry, business and the military1. In particular, the utilization of U in the military and nuclear industry has resulted in a plethora of health risks2. Exposure to metals such as U dysregulates the immune system due to oxidative stress and apoptosis, as well as altered expression of key regulators of the immune system3. Furthermore, the presence of autoantibodies against native DNA and/or chromatin has been associated with elevated U intake from drinking water4. Uranyl acetate, a by-product of U enrichment procedures, mostly comes from fuel used in nuclear power plants. The potential for nuclear conflicts to escalate in certain regions, coupled with the rising demand for nuclear fuel, gives rise to concerns about the possibility of uranyl acetate leakage into nature5,6. The capacity of uranyl acetate to enter the body through air, water, and food, its accumulation in the food chain, the chemical and radiological hazards it presents, and the side effects of chelation therapies combine to make it a serious candidate for multiple toxicity6. The discovery of side-effect safe biological natural resources to reduce the destructive effects of ecological threats such as uranyl acetate on living organisms has long been of interest to researchers.Abelmoschus esculentus (L.) Moench (Hibiscus esculentus L.), commonly known as okra or lady’s finger, is a vegetable species belonging to the Malvaceae family, with origins in Ethiopia and Sudan7,8. It comes from the Arabic phrase “Abual-misk,” which means “Father of Musk,” and “Kaab-el-misk,” which refers to the musky smell of the plant’s seeds9. It is known by a number of intriguing names, including “plant viagra” in the USA and “green pana” in Japan, due to its functional qualities10. It is cultivated in tropical and subtropical regions worldwide7,8. In several nations, including Türkiye, Cyprus, India, Bangladesh, Thailand, Pakistan, Afghanistan, Myanmar, Malaysia, Brazil, Ethiopia and the Southern United States, A. esculentus plants are farmed for commercial purposes11. A. esculentus L. has a variety of applications for its leaves, flowers, buds, stems, pods and seeds and is regarded as a highly versatile crop12. In Turkey, the leaves of the plant are utilized in the manufacture of pharmaceuticals, and the mature seeds are ground and incorporated into the preparation of coffee and soup7. The fruits are also traditionally consumed directly as a stewed dish. It has been reported that A. esculentus extract contains significant levels of phenols and flavonoids, which have been demonstrated to confer effective antihemolytic, antioxidant, antimicrobial and antihypoxic properties to the plant8,9. A. esculentus is also an important source of carotenoids, tocopherols and minerals and is particularly rich in vitamins A, E and C, as well as sodium, potassium, calcium and magnesium9. It has been demonstrated that extracts of A. esculentus, which are abundant in phenolic compounds, such as quercetin and rutin, can effectively mitigate the adverse effects of stress factors, including UV-B radiation, by alleviating cytotoxicity, oxidative stress, and DNA damage13.The onion (Allium cepa L.) is a vegetable that offers a substantial nutritional profile and economical production while also providing a unique subject for cytogenetic investigations due to its fast-growing roots and easily traceable, large-sized chromosomes14. In addition to working in tandem with the Comet test to predict DNA damage caused by contaminants, the Allium test has an over 82% correlation with studies on mammals15,16. The Comet assay, often known as single cell gel electrophoresis, has been one of the principal cytogenetic methods for analyzing DNA lesions such as strand breaks and repair processes in eukaryotic cells for about 40 years17.In the present study, a comprehensive approach was used to investigate the uranyl acetate-induced toxicity and the antitoxic potential of A. esculentus extract in the model plant A. cepa. For this purpose, some various physiological, cytogenetic and biochemical parameters were investigated. These included rooting percentage, root elongation, weight increase, mitotic index (MI), micronucleus (MN) formation, chromosomal aberrations (CAs), lipid peroxidation [malondialdehyde (MDA) level], superoxide dismutase (SOD) enzyme activity, catalase (CAT) enzyme activity, chlorophyll a and chlorophyll b levels. The experiments were conducted using individual applications and mixtures of uranyl acetate and A. esculentus. Cross-sections of the roots were analyzed in order to determine the extent of damage to meristematic cells, and the percentage of DNA tails obtained from the Comet assay was used to determine the level of DNA damage.Materials and methodsMaterialsA. cepa bulbs of similar size employed as test organisms in the study were procured from a local marketplace in Giresun (Türkiye) province. The bulbs were cultivated in the same year as the study without the use of chemical agents. The uranyl acetate dihydrate (UO2(CH3COO)2 ⋅ 2H2O) (CAS number: 6159-44-0) with ≥ 98 purity, employed in the experiments and analyses, was obtained from Sigma-Aldrich. All other chemicals utilized in this study are of analytical grade. The dried flowers of A. esculentus, which were employed in the study, were procured from “Aşçı Baharatları” a commercial supplier based in Ordu, Turkey. The flowers were ground into a fine powder using an electric grinder. Ground floral (1 g) powder was extracted with 100 mL of ethanol for 48 h on a magnetic stirrer. Ethanol was evaporated after filtration (Whatman no. 4) with a rotary evaporator (Heidolph, Hei-VAPML, Germany) operating under vacuum. Ethanol was selected as the solvent of choice due to its widespread and successful use in the preparation of similar herbal extracts and its status as the safest option for living organisms in comparison to other solvents. Following this, the extract was stored in a refrigerator until it was diluted with water in order to prepare the test solutions.Experimental setupThe doses of uranyl acetate dihydrate employed in the study were based on the doses previously mentioned by Aydın et al.18. In the referenced study, 0.1 mg mL⁻¹ uranyl acetate was found to reduce the germination percentage in A. cepa to 46%, yet root elongation remained sufficient to allow for sample collection for further analyses. The concentrations of A. esculentus extract selected for the study are in accordance with the doses showing protective effects reported by Alqasoumi19. In the aforementioned study, the antioxidant and protective performance of the extract at the specified doses was found to be similar to that of the reference standard. Six groups of 50 onions each were formed, with one group serving as control (Table 1). The bulbs were positioned in sterile glass beakers, with the root plates in contact with the respective solutions. The onion bulbs were kept at room temperature and in the dark for three days, with the solutions being replenished on a daily basis. The harvesting of the bulbs was conducted at the conclusion of the third day. For chlorophyll analyses, the aforementioned procedure was continued for six days. The harvested bulbs were subjected to the analyses presented in Fig. 1. Experimental research on plants, including the procurement of plant material, complies with institutional, national and international guidelines and legislation20.Table 1 Treatment groups and their corresponding test solutions used in the A. cepa bioassay.Full size tableFig. 1Diagrammatic representation of experimental design and analytical parameters used for investigating the mitigative potential of A. esculentus extract against uranyl acetate toxicity.Full size imagePhysiological analysesThe effect of uranyl acetate and A. esculentus extract treatments on the rooting percentages of A. cepa bulbs was calculated using the equation (Eq. 1) of Atik et al.21 on fifty bulbs from each group (n = 50).$${\text{Rooting percentage (\% ) = Number of rooted bulbs/Total number of bulbs }} \times {\text{100}}$$(1)Root elongation and weight gain were determined in 10 bulbs that were randomly selected from the rooted bulbs for each group. Root elongation was assessed by measuring the length of roots using a digital compass (n = 10). Weight increase was examined by weighing ten bulbs before and after application with the aid of a precision balance (n = 10).Cytogenetic analysesPrior to investigating the impact of uranyl acetate and A. esculentus extract treatments on cytogenetic effects, the roots were carefully rinsed with distilled water to eliminate any residual chemical substances. The frequency of CAs, MNs, and the MI value were determined from the same preparations. Mitotic slides were prepared according to the method suggested by Staykova et al.22 with minor modifications. Approximately 1 cm-long pieces cut from the tips of the roots were fixed in Clarke solution (three volumes of ethanol and one volume of glacial acetic acid) to fix them for two hours. The fixed root tips were subjected to hydrolysis in 1 N HCl in a hot water bath at 60 °C for 14 min, after which they were washed with glacial acetic acid (45%). The hydrolyzed root tips were subjected to a 16-hour staining process with aceto-carmine (1%), after which they were crushed in a drop of 45% between the slide and the coverslip. This solution was then imaged at a magnification of ×500 under an IRMECO IM-450 TI research microscope. MN and CAs were determined by examining 1,000 cells in each group (n = 10), whereas MI was determined by reviewing 10,000 cells in each group (n = 10). The mitotic index (MI), which represents the ratio of mitotic cells to all cells, was calculated using the following (Eq. 1)8:$${\text{MI = Number of cells undergoing mitosis/Total number of cells }} \times {\text{100}}$$(2)Comet assayThe method proposed by Sharma et al.23 was used to obtain the DNA of A. cepa root required for the Comet test. The Comet test was conducted in accordance with the methodology proposed by Dikilitaş and Koçyiğit24. The slides to be used in the Comet test were sterilized in ethyl alcohol for one day and then oven dried. Then, 100 µL of agarose was layered on the slide with 1% normal melting point (NMP) agarose. The gel on the slides was allowed to freeze rapidly at 4 ºC for 5 min. A 100 µL of mixture containing one part cell suspension and seven parts low melting point (1%) agarose was applied as a second layer on a slide at 40 °C and immediately covered with a coverslip. Following a five-minute refrigeration period and the removal of the coverslips, the preparations underwent a 40-minute incubation in buffer solution within the electrophoresis tank. The preparations were then electrophoresed for 20 min at 86 V cm− 1 (20 V, 300 mA). Following the neutralization process using tris-buffer, the slides were stained with 100 µL of ethidium bromide (0.2 mg mL− 1) and analyzed using a fluorescent microscope. Utilizing “TriTek 2.0.0.38 Automatic Comet Assay” software, the obtained pictures were examined. A percentage (%) was used to indicate how many DNA fragments were included in the head and tail sections. To assess the degree of DNA damage caused by the treatments, the scale developed by Jayawardena et al.25 was used. The degree of DNA damage, expressed as quantitative data, was calculated using the scale originally proposed by Pereira et al.26, with particular consideration of the percentage of tail DNA.Biochemical analysesThe methodology proposed by Unyayar et al.27 was employed to quantify MDA levels, which serve as a marker for lipid peroxidation induced by oxidative stress. For this purpose, 0.5 g of freshly harvested root tips were subjected to mechanical homogenization in a solution of 1 mL 5% trichloroacetic acid. Equal volumes of supernatant, thiobarbituric acid (5%) and trichloroacetic acid (20%) were mixed in and the contents were allowed to react at 96 °C for 30 min in a new tube. An equal volume of the supernatant was combined with an equal volume of thiobarbituric acid (5%) and trichloroacetic acid (20%), and the resulting mixture was allowed to react in a new tube for 30 min at 96 °C. Subsequently, the reaction was terminated by submerging the tube in an ice bath. The mixture underwent centrifugation at 10,000 g for five minutes, after which the absorbance of the supernatant was determined at 532 nm. The MDA level is expressed in micromoles of MDA per gram of fresh weight (µM g− 1 FW).The procedure recommended by Zou et al.28 was employed for the preparation of enzyme extracts for subsequent measurement of SOD and CAT enzyme activity. Accordingly, 0.5 g of fresh root tip sample was homogenized in 5 mL of monosodium phosphate buffer (50 mM, pH 7.8) and then centrifuged at 10,500 g for 20 min at 4 °C. The obtained supernatant was kept in a refrigerator at 4 °C until the activity of the enzymes SOD and CAT were measured.To measure the activity of the SOD enzyme, a reaction solution comprising 1.5 mL of 0.05 M monosodium phosphate buffer, 0.3 mL of 130 mM methionine, 0.3 mL of 750 µM nitroblue tetrazolium chloride, 0.3 mL of 0.1 mM EDTA, 0.3 mL of 20 µM riboflavin, 0.01 mL of enzyme extract, 0.01 mL of 4% insoluble polyvinylpyrrolidone, and 0.28 mL of de-ionized water were prepared. The reaction process was initiated by exposing the tubes to two 15-watt fluorescent lamps for 10 min. Following this, the reaction was terminated by transferring the tubes to a dark environment for a further 10 minutes29. The absorbance was determined at 560 nm, and the SOD activity was expressed as units per milligram of fresh weight (U mg− 1 FW).The activity of the CAT enzyme was determined by measuring the decrease in absorbance resulting from the consumption of hydrogen peroxide. This was accomplished by adding 0.2 mL of enzyme extract to 2.8 mL of reaction solution, which was composed of 0.3 mL 0.1 M hydrogen peroxide, 1.0 mL distilled water, and 1.5 mL 200 mM monosodium phosphate buffer. The alteration in absorption caused by the enzymatic reaction was measured at a wavelength of 240 nm using a UV-VIS spectrophotometer at 25°C30. CAT enzyme activity was expressed as OD240 nm min g− 1.The chlorophyll content of A. cepa leaves from the experimental groups was determined by modifying the method proposed by Kaydan et al.31. For this purpose, 0.1 g of fresh leaf sample was pulverized with a plastic stick in a glass tube containing 2.5 mL of 80% acetone and then stored in the dark for seven days. This procedure was conducted rapidly and at low temperature to prevent the acetone from evaporating. Following a seven-day incubation period, the mixture was filtered. Prior to centrifugation at 1,000 g, an additional 2.5 ml of 80% acetone was added and thoroughly mixed. The absorbance of the obtained supernatant was then measured spectrophotometrically at both 645 and 663 nm wavelengths. The equation presented by Witham et al.32 (Eqs. 3, 4) was used to compute the concentrations of chlorophyll a and chlorophyll b pigments.$${\text{Chlorophyll a}} = \left( {{\text{12}}.{\text{7 }} \times {\text{ A}}_{{{\text{663}}}} - {\text{2}}.{\text{69 }} \times {\text{ A}}_{{{\text{645}}}} } \right) \times \left( {{\text{V}}/{\text{1}}000 \times {\text{W}}} \right)$$(3)$${\text{Chlorophyll b}} = \left( {{\text{22}}.{\text{9}} \times {\text{A}}_{{{\text{645}}}} - {\text{4}}.{\text{68}} \times {\text{A}}_{{{\text{663}}}} } \right) \times \left( {{\text{V}}/{\text{1}}000 \times {\text{W}}} \right)$$(4)A663 represents the absorbances of the supernatant at 663 nm wavelengths, V represents the final volume of 80% acetone with the supernatant (mL), W represents the weight of the fresh leaf (g) and A645 represents the absorbances of the supernatant at 645 nm wavelengths.Meristematic tissue analysisTo examine the protective effect of A. esculentus extract against uranyl acetate-induced meristematic tissue damage, cross-sections of the roots of A. cepa bulbs in UA group were taken. Cross-sections were imaged using a digital research microscope (IM-450 TI) for examination after being stained with a drop of 1% methylene blue and covered with coverslips. Cross-sections were imaged using a digital research microscope (IM-450 TI) for examination after being stained with a drop of 1% methylene blue and covered with coverslips. A total of one hundred images from ten roots per group were analyzed and graded to assess the extent of meristematic tissue damage. The observed damages were categorized according to the frequency of meristematic tissue damage types for the groups, as follows: (-): 0–10 normal appearance, (+): little damage, (++): moderate damage, (+++): severe damage.Phenolic content analysisThe phenolic composition of A. esculentus was determined through the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For this purpose, a sample of A. esculentus powder (1 g) was extracted using methanol-dichloromethane (4:1), filtered using a syringe with a 0.45 μm filter and subsequently analyzed. The analysis was performed using an LC-MS/MS instrument (Thermo Scientific) using an ODS Hypersil 4.6*250 mm column at a flow rate of 0.7 mL min− 1 for 34 min at 30°C33,34. The LC-MS/MS analysis was conducted at Hitit University Scientific Technical Application and Research Center (HUBTUAM). All conditions used for chromatographic separation and analyte detection and the total ion chromatogram are given in the supplementary file.Statistical analysisStudy results were subjected to statistical analysis using the IBM SPSS Statistics 23 software. The data were expressed as mean ± standard deviation as a consequence of the analysis. The data demonstrated a normal distribution, as indicated by both the Shapiro-Wilk and Kolmogorov-Smirnov tests (p > 0.05). Furthermore, the Swekness-Kurtosis values of the data are within the range that is suitable for normal distribution (-1, + 1). Therefore, parametric tests were used for statistical analysis. Since it was seen by Levene’s test that the variances were homogeneously distributed, one-way ANOVA and Duncan’s test were applied to understand the statistical difference between the groups. A p-value of less than 0.05 was deemed to be statistically significant.Results and discussionThe growth-related physiological effects induced by uranyl acetate and A. esculentus extract are presented in Table 1. Bulbs with roots reaching a length of 1 cm or more during the experimental period were classified as ‘rooted’. Control group and AE 1 and AE 2 groups treated with doses of A. esculentus extracts exhibited almost 100% rooting. These findings suggested that A. esculentus extract solutions did not induce any toxic effects on root emergence in A. cepa bulbs. In addition, the administration of A. esculentus extracts did not result in any adverse effects on the other selected physiological parameters. Indeed, the root elongation and weight increase values of AE 1 and AE 2 groups were not statistically different from those of control group, according to the results of the statistical analysis (Table 2). Conversely, the percentage of rooting observed in UA group treated with uranyl acetate decreased to 43%. Furthermore, the root elongation and weight increase values of the rooted A. cepa bulbs exhibited a marked decline, with a reduction of 85% and 77%, respectively, in comparison to control group. The results of this study corroborate those of Aydın et al.18, which indicated that uranyl acetate is a growth retardant for A. cepa. However, U, which lacks a clearly defined biological function, has been reported to exert genotoxic effects in plants, impair seed germination, induce oxidative stress, inhibit growth, nutrient uptake and photosynthesis35. Additionally, numerous studies have demonstrated that other heavy metals impede plant growth, as evidenced by the experiments conducted on A. cepa and other plant species36,37,38. The uptake of water and minerals and the damage to cellular membranes in meristematic regions, along with the processes of cell division and elongation, have an influence on root growth in plants39. One of the factors that amplifies these effects and retards growth in A. cepa exposed to uranyl acetate may be the reactive oxygen species (ROS)-induced oxidative stress that the plant is compelled to combat during heavy metal exposure. The growth parameter values observed in UAAE 1 and UAAE 2 groups, where uranyl acetate and A. esculentus extract had been applied in a combined mixture, demonstrated elevated values in comparison to UA group values (Table 2). Moreover, the amelioration of uranyl acetate-induced growth inhibition in A. cepa became more pronounced as the concentration of A. esculentus in the uranyl acetate + A. esculentus mixture increased. The mean rooting percentage values for UAAE 1 and UAAE 2 groups were found to be 57% and 70%, respectively. Additionally, the root elongation values in UAAE 1 and UAAE 2 groups were 1.2 and 2.5 times more than UA group, respectively. Furthermore, the mean weight increase of UAAE 1 group was 0.9 times more than UA group, while the mean weight increase of UAAE 2 group was 1.8 times more than UA group. There was no prior research comparing the preventive effect of A. esculentus against uranyl acetate-induced growth retardation in A. cepa or other plants. However, Salvia officinalis extract, previously tested against uranyl acetate-induced U toxicity in A. cepa, was found to reduce the loss in germination percentage, root elongation and weight gain18. In addition, research has demonstrated that the okra plant is capable of acting as a heavy metal chelator, thereby facilitating phytoremediation40. The accumulation of phenol compounds in plants following heavy metal stress has been demonstrated to alleviate oxidative stress by chelating metal ions. Consequently, these compounds may reduce uranyl acetate-induced physiological toxicity in A. esculentus40,41. It is also possible that the A. esculentus extract reduces growth retardation by limiting the uptake and transport of uranyl acetate from the root medium, which may be due to its ability to reduce damage to cell membranes.Table 2 Effects of uranyl acetate and A. esculentus extract on selected physiological parameters.Full size tableThe cytogenetic parameter results of control, AE 1 and AE 2 groups treated with tap water, 250 mg L− 1A. esculentus extract and 500 mg L− 1A. esculentus extract, respectively, did not exhibit a statistically significant difference (p > 0.05) (Table 3). Then, the applied doses of A. esculentus did not disturb the cytogenetic pattern of A. cepa root meristem cells. However, exposure to uranyl acetate induced a marked cytotoxicity in the members of UA group (Fig. 2; Table 3). The mean MI value of UA group exhibited a 37% reduction in comparison to control group. Moreover, the values of MN and CAs in UA group were found to be significantly higher than those observed in control group (p