INTRODUCTIONNew approaches to food testing are becomingincreasingly urgent today, in view of continuouslygrowing production and consumption of various foods.These approaches are primarily meant to ensure foodsafety by identifying possible toxic effects that foodproducts and related additives may have on humanhealth [1–4]. Any food component can have a negativeeffect on the human body. Excessive consumption canlead to the accumulation of toxic metabolic products.Some components can cause allergic reactions andmodulate adaptation reactions [5]. Such studies areprimarily based on in silico and in vivo methods oftesting various types of food products.Fruit juice is an integral part of the human dietand, undoubtedly, a complex food system. It containsphysiologically active substances (vitamins, minerals,antioxidants, enzymes, and amino acids) that regulate avariety of metabolic processes and increase the body’sresistance to infections. In addition, epidemiologicalstudies have proved that fruits and vegetables reducethe risk of chronic diseases [6, 7]. Clinical studies also confirm that fruit juice can have beneficial effects onblood parameters, cholesterol, and heart function, aswell as prevent cancer and Alzheimer’s disease [8–11].However, the benefits of fruit juice are not asevident as they may seem [12]. As we know, naturalmutagens, such as pyrolysidine alkaloids and someflavonoids, account for about 1% of dry matter in almostall higher plants. Moreover, vitamins C, E, and A canhave mutagen-potentiating effects [13]. Recent studieshave shown that fruits and juices can contribute to thedevelopment of cancer and asthma in children [7, 14–16].Sugars contained in fruit juice and their potentiallyadverse metabolic effects have long been in the centerof scientific debates. Fructose, in particular, is one of themain carbohydrate components of fruit juice. As earlyas the 1980s, it was considered responsible for severalmetabolic abnormalities [17, 18]. This carbohydrate canbe “toxic”, especially when consumed with sweeteneddrinks. Moreover, it can participate in the pathogenesisof noncommunicable diseases such as obesity, diabetes,or arthritis [19, 20]. Sucrose, another carbohydratecomponent of fruit juice, has also shown negativemutagenic effects [21]. In the USA and Europe, a halfof sugar consumption accounts for sweetened productswith a thick consistency (yogurt, candy and chocolatebars, ice cream, etc.) and the other half, for sweetenedfizzy drinks and fruit juices. The negative health effectsof fructose have encouraged European countries toimpose taxes on sweetened drinks [22].Quality control is an equally important aspectof fruit juice safety. The past decades have seen asignificant increase in the demand for juice, partlydue to continuous improvement of its sensory (color,smell, texture, and taste) and technological (convenientpackaging, long shelf life) characteristics. As aresult, juice composition has undergone a numberof changes, with added microelements and syntheticsubstances (acidity regulators, stabilizers, thickeners,and sweeteners). The technology of juice production(e.g., heat treatment) also affects juice properties.Although the use of these additives is strictly regulated,scientists are increasingly emphasizing a need forrigorous research into the mechanisms of their toxicmanifestations [23, 24].Studies have shown that food additives can leadto cancer and change the functioning of variousorgans [25–27]. Children are especially vulnerable totheir toxic effects that can provoke allergies and otherdiseases if manufacturers do not follow strict regulations[28]. Although several types of food additives canbe used in juice in various combinations, there havebeen no studies into their integrated toxic effect on thehuman body. Moreover, as chemically active agents,these additives or their oxidation products can interactwith natural organic or inorganic juice compounds andcause especially dangerous mutagenic and carcinogeniceffects [29].In this regard, in vivo studies of subchronic toxicityof fruit juice components are becoming increasinglyurgent. Modern food scientists aim to develop modelsin which the processes of detoxification and metabolismof toxic compounds are similar to those in the humanbody. At the same time, they strive not to use laboratoryanimals [4].We find biotesting quite effective when usingplants, in particular Allium cepa roots (Allium test).This test has been successfully used to study toxicity,cytotoxicity, and genotoxicity of various agents,including food additives, as well as to determinegenotoxic effects of medicinal plant extracts [23,30–32]. The Allium test is simple, economical, wellreproducible, highly sensitive, applicable in a wide pHrange (3.5–11.0), and just as efficient as other biotests.We believe that this test can be reliably used to assesssubchronic toxic effects of various juice components,both individually and in combination with each other.Similar studies in animals may not produce objectiveresults. The components under study may be present inthe animals’ basic diet, compromising the results.Our aim was a comparative study of subchronictoxic effects that apple juice, its components, andethanol have on biomass growth, oxidative enzymeactivity at the cellular level, as well as the nature andfrequency of proliferative and cytogenetic disorders inAllium cepa roots.STUDY OBJECTS AND METHODSTo model the composition of apple juice, we used thefollowing materials: glucose (SIGMA-ALDRICH, lot.SLBZ9363, Germany), fructose (SIGMA-ALDRICH, lot.SLCC1647, Germany), sucrose (SIGMA-ALDRICH, lot.BCCB2955, Germany), D-sorbitol (SIGMA-ALDRICH,lot.BCBT4918, Germany), malic acid (SIGMAALDRICH,lot.MKBS7851, Germany), and clarifiedapple juice (10.5% carbohydrates) purchased from aretail outlet.For biotesting, we used small 5–7 g Allium cepa L.onions of Stuttgart variety with a diameter of 2.5–3 cm,with their dry scaly outer layers removed. The rootswere preliminarily germinated in 15 cm3 test tubes withbottled water in a thermostat (23–25°C) for two or threedays in complete darkness. The bulbs with a sproutedroot length of at least 1 cm were selected for furtherexperiments. Prior to treating them with juice solutionsand other compounds listed above, we measured theaverage root mass in the control group.Then, the control samples were incubated in water,while the test samples were incubated in aqueoussolutions in a thermostat (23–25°С) in completedarkness for 1, 2, or 3 days, depending on the purposeof the experiment. After incubation, the roots were cutoff, wiped with filter paper, and weighed [33]. EC50 wasdetermined as a concentration of material that reducedthe test function (growth in root mass) by 50% compared323Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328to the control, taking into account the average massof the roots before treatment (except when they weretreated with fructose).For cytogenetic analysis, the cells of the root apicalmeristem were stained with a 2% aceto-orcein solution(1 g of orcein diluted in 50 cm3 o f 4 5% C H3COOH).For long-term storage, the roots were placed in a 70%ethanol solution used as a preservative. Instant squashpreparations were obtained to analyze the divisionof apical meristem cells, using an Axioskop 40 lightmicroscope (Zeiss). In particular, we determined themitotic index (ratio of dividing cells to total cells)and the chromosome aberration index (number ofchromosomal aberrations related to total cells).The intensity of lipid peroxidation in root tissues wasdetermined based on the amount of malondialdehyde(MDA) interacting with 2-thiobarbituric acid andexpressed in μmol/g (MDA in fresh mass) [34]. Weplaced 0.2–0.9 (± 0.0001) g into a 15 cm3 polymer tube,added 1 cm3 of trichloroacetic acid (Merck, Germany) ata concentration of 200 g/dm3 and then another 3 cm3 ofthe same solution after stirring the mixture. The tubeswere centrifuged at 1000 g and 4°C for 15 min. Then,we transferred 1 cm3 of the upper liquid layer intoanother tube and added 4 cm3 of thiobarbituric solution ‒0.5 g thiobarbituric acid (Diaem, Russia) and 100 cm3 oftrichloroacetic acid (200 g/dm3). The tubes were tightlyclosed and placed for 30 min in a water bath at 95°C,followed by cooling in an ice bath. Next, the tubes werecentrifuged for 10 min at 1000 g and 20°C. The solutionswere spectrophotometrically detected on a CaryWinUV 100 spectrophotometer (Varian, USA) at 600and 532 nm.Statistical processing was performed in MicrosoftExcel and Statistica (v. 12). The Student’s criterionand Fisher transformation were used for comparativeanalysis of percentages.RESULTS AND DISCUSSIONAfter a three-day sprouting, the onion roots weretreated with apple juice diluted with water for 2 daysto determine the degree of juice dilution that causessubchronic toxicity. According to the Allium test,toxicity was determined by the changes in root massafter exposure to juice solutions, compared to thecontrol. As we can see in Fig. 1, a decrease in rootmass was observed at ten times dilution and EC50 wasrecorded at five times dilution (P ≤ 0.15).The cytological analysis of the root meristem cellsshowed that higher juice concentrations decreasedthe mitotic index more intensively (Fig. 2) than thegrowth in root mass (Fig. 1). As we can see, the level ofproliferation for meristematic cells, when treated with a1:20 diluted solution of apple juice, was half the controlvalues, and their division almost stopped in the rootswith a 50% delay in mass growth (EC50).As we know, plants grow due to two main processes,cell division and extension. Like all eukaryotes,plant cells enter the cell cycle in response to externalmitogenic stimuli. This process is regulated by a largenumber of compounds, such as phytohormones, ARGOSproteins, CLE peptides, transcription factors, cyclins,and cyclin-dependent protein kinases. Decreased cellproliferation during stress or after treatment withabscisic acid may result from activated expression ofgenes that encode protein inhibitors of cyclin-dependentprotein kinases, ICK/KRP. However, the mechanismsthat control differentiation can function independentlyof the cell cycle [35]. It appears that the subchronicamounts of apple juice triggered similar processes in ourstudy and, therefore, the inhibition of cell proliferationdid not significantly affect the growth in root mass.The percentage of chromosomal aberrations individing cells in relation to total stained cells was quitelow, about 0.4%, both in the control and the test samplestreated with 1:20 and 1:10 diluted juice. We found noeffect dependent on the amount. Neither could wedetermine this indicator in the test samples treated witha higher concentration of juice (1:5 and 1:2 dilution) dueto the absence of dividing cells.The abnormalities detected in both the control andthe test samples included the adhesion of chromosomesto each other, their leading during anaphase, as wellFigure 1 Growth in root mass after treatment with apple juicein different concentrations (n = 10). *P ≤ 0.15controljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**controljuice 1:20juice 1:10juice 1:5juice 1:20246810Mitotic index, %020406080100120Growth in root mass,% of controlcontrolfructose2 %fructose5 %45678 index, %10152025MDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**controljuice 1:20juice 1:10juice 1:5juice 1:2246810Mitotic index, %020406080100120Growth in root mass,% of controlcontrolfructose2 %fructose5 %345678 index, %510152025MDA concentration,μmol/g of fresh massFigure 2 Mitotic index of root meristem cells after treatmentwith apple juice in different concentrations (n = 10). *P ≤ 0.05controljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**0246810Mitotic index, %controlfructose2 %fructose5 %345678Mitotic index, %510152025MDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control*controljuice 1:200246810Mitotic index, %controlfructose2 %fructose5 %5678index, %10152025MDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**controljuice 0246810Mitotic index, %controlfructose678%152025concentration,fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**control0246810Mitotic index, %78152025concentration,fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**10Mitotic index, %78concentration,fresh mass324Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328controlfructose2 %fructose5 %fructose10 %fructose15 %01234567Mitotic index, %as disorganization and disordered separation duringmetaphase and anaphase. However, these abnormalitieswere not distributed evenly among the samples. Forexample, aberrations (Fig. 3) and anaphase leadingwere almost ten times as high in the test samples.Also, micronuclei were detected during telophaseand interphase in the samples treated with tenfolddiluted juice.With the data at hand, we had to understand which ofthe juice components was responsible for the identifiedtoxic effects and to what extent. Carbohydrates area major component of apple juice, with up to 10% offructose, glucose, and sucrose (in 100 g juice). Takinginto account published data on the negative effects ofglucose on plant growth and development, we conductedseveral experiments to determine their toxicity for onionroots [36, 37]. We started with fructose, as its content inapple juice is two times as high as that of glucose andsucrose.As we can see in Fig. 4, higher concentrations offructose delayed the growth in root mass, but onlya 10% concentration of this carbohydrate revealed asignificant difference. After treatment with 10 and 15%fructose solutions, the roots died, becoming thin, soft,and slightly mucous. In the Allium test, this findingprobably indicated acute toxicity of fructose in the givenconcentrations. Thus, the concentration of fructoseshould not exceed 10%.Fructose at concentrations of 2 and 5% decreasedthe mitotic index in the test samples by only 17 and33%, respectively, compared to the control (Fig. 5).Like in the previous test, the comparative cytogeneticanalysis did not reveal a significant increase in thenumber of chromosomal aberrations, compared to thecontrol. However, we observed some redistribution intheir spectrum. For example, higher concentrations offructose in the test samples caused more disorders suchas chromosomal bridges, fragmentation, and segregation(up to 20%), compared to the control.According to the results, the subchronic toxicity offructose, one of the main components of apple juice, ismainly associated with a weak mitosuppressive effect inthe root meristem cells.Then, we prepared a model aqueous solution fromthe main chemical components of apple juice. Theirconcentration ratios corresponded to those in juice [38].In particular, 100 mL of the model solution contained7 g fructose, 2 g glucose, 1 g sucrose, 0.5 g D-sorbitol,and 0.3 g malic acid. Prior to that, we had measuredthe pH of the study objects to make sure that its rangewas acceptable for the Allium test (Table 1). Next, weanalyzed the subchronic toxicity of the resulting modelsolution and apple juice in Allium cepa roots after twodays of germination and two days of treatment.According to the results, the growth in root massafter treatment with juice was 40% lower than afterusing the model solution (Table 2), despite the samedegree of dilution (1:5, P < 0.05). The cytologicalanalysis showed that the mitotic index of the rootmeristem cells after treatment with the 1:10 and 1:5model solutions did not differ much from the control.However, treatment with the 1:10 and 1:5 diluted juice,just like in the previous experiment (Fig. 2), reducedthe mitotic index ten times and led to an almostcomplete halt in cell division. Thus, the chemical05101520Control 1 %ethanol2 %ethanolJuice 1:2 Juice1:2+1%ethanolJuice1:2+2%ethanolMDA concentration,μmol/g of fresh massFigure 3 Chromosomal aberrations in onion root meristemcells: adhesion in metaphase (a), leading in anaphase (b),disorganization in anaphase (c), and a micronucleus intelophase (d)05101520MDA concentration,μmol/g of fresh massControl Juice 1:10 Juice 1:5 Modelsolution 1:10Modelsolution 1:50510152025Control 1 %ethanol2 %ethanolJuice 1:2 Juice1:2+1%ethanolJuice1:2+2%ethanolMDA concentration,μmol/g of fresh mass(a) (b)(c) (d)Figure 4 Growth in root mass after fructose treatment(n = 7). *P ≤ 0.1juice 1:5juice 1:2**controljuice 1:20juice 1:10juice 1:5juice 1:20246810Mitotic index, %control fructose2 %fructose5 %fructose10 %fructose15 %020406080100120Growth in root mass,% of controlfructose10 %fructose15 %0510152025Control Juice 1:10 Juice 1:5 Modelsolution 1:10Modelsolution 1:5MDA concentration,μmol/g of fresh massJuice 1:2 Juice1:2+1%ethanolJuice1:2+2%ethanolcontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**control0246810Mitotic index, %controlfructose2 %fructose5 %fructose10 %fructose15 %-1012345678Mitotic index, %0510152025MDA concentration,μmol/g of fresh mass0510152025Control 1 %ethanol2 %ethanolJuice 1:2 Juice1:2+1%ethanolJuice1:2+2%ethanolMDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**0246810Mitotic index, %controlfructose2 %fructose5 %fructose10 %fructose15 %-1012345678Mitotic index, %0510152025MDA concentration,μmol/g of fresh mass0510152025Control 1 %ethanol2 %ethanolJuice 1:2 Juice1:2+1%ethanolJuice1:2+2%ethanolMDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**Mitotic index, %controlfructose2 %fructose5 %fructose10 %fructose15 %-1012345678Mitotic index, %MDA concentration,0510152025Control 1 %ethanol2 %ethanolJuice 1:2 Juice1:2+1%ethanolJuice1:2+2%ethanolMDA concentration,μmol/g of fresh massFigure 5 Mitotic index of root meristem cells after fructosetreatment (n = 7). *P ≤ 0.05controljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**control0246810Mitotic index, %controlfructose2 %fructose5 %45678index, %510152025MDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**controljuice 1:20juice 0246810Mitotic index, %controlfructose2 %fructose5 %fructose10 %fructose12345678Mitotic index, %0510152025MDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120 Growth in root mass,% of control**controljuice 0246810Mitotic index, %controlfructose2 %fructose5 %345678Mitotic index, %510152025MDA concentration,μmol/g of fresh masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**control0246810Mitotic index, %82025concentration,masscontroljuice 1:20juice 1:10juice 1:5juice 1:2020406080100120Growth in root mass,% of control**Mitotic index, %8concentration,mass325Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328components of the model solution, which make upthe bulk of juice solids, were not responsible for thesubchronic toxicity associated with violation of mitosisin the roots. Obviously, this effect was caused by otherjuice compounds with antiproliferative activity ofnatural origin.Based on the data, we can conclude that thesecompounds (one or more) are present in juice in smallquantities and have high biological activity. We needfurther studies to identify these substances and betterunderstand the mechanisms of potential juice toxicity.MDA is known to reflect the degree of lipidperoxidation resulting from the oxidation process. Thehigher its concentration, the more damaged are lipidsin the walls of plant cells. In our study, the treatment ofonion roots with the 1:10 and 1:5 diluted model solutionsproduced a dose-dependent increase in MDA, with itsmaximum levels twice as high as in the control samples(Fig. 6). However, apple juice in the dilutions of 1:10 and1:5 increased this indicator by only 11%. Apparently,these results are indicative of the juice’s antioxidantactivity.To study toxic effects, we treated the onion roots,which had germinated for two days, with the 1:10 and1:2 diluted juice for only one day. As we can see inTable 3, a day of incubation brought about a slightlyhigher (10%) decrease in mitotic indices in these testsamples than in those treated for two days, compared tothe control (Fig. 2). Thus, the toxic effect was recordedas early as after the first cycle of cell division, whilethe decrease in root mass growth was more likely to becumulative.Food additives are commonly studied for toxicityseparately from those food products which they arepart of. We believe that such practice does not allowscientists to objectively determine the patterns of toxicmanifestations. Therefore, our further experimentsattempted to evaluate the effect of ethanol on thepreviously detected toxicity of Allium cepa roots, whichhad been germinated for two days and then incubatedwith apple juice for another two days. We chose thisfood additive due to the fact that 1 and 2% aqueousethanol solutions delay the growth of Allium cepa rootswithin EC50 [29, 32]. In addition, ethanol may be part ofsome juice-containing products.Table 4 shows that 1% ethanol increased the averagemass of the roots treated with the 1:2 diluted juiceby a factor of five. We believe that this effect can beassociated with the activity of lipid oxidation enzymesand its regulation. Indeed, raising ethanol concentrationto 2% not only decreased their activity, but made itlower than the control values (Fig. 4). However, in the1:2 diluted juice samples, MDA was almost 1.7 times ashigh as in the control (Fig. 7), which we had expectedfrom the previous results (Fig. 3). Thus, 1% ethanolappeared to slow down the destruction of cell walllipids caused by the juice components, which had apositive effect on the root growth. The question is, whyis it that a higher concentration of ethanol (2%) did notcause a similar effect? Probably, despite lower lipidoxidation, the total toxicity of 2% ethanol was so highthat it prevented the roots from growing and developing.Table 1 pH of study objectsStudy object pHJuice 3.88Model solution 2.62Juice:water 1:5 4.23Juice:water 1:10 5.11Model solution:water 1:5 3.78Model solution:water 1:10 4.63Table 2 Root mass growth, mitotic activity, and chromosomalaberrations of onion root meristem cells after two daysof treatment with apple juice and model solution (n = 10)Test variant Growth inroot mass,g/onionMitoticindex, %Chromosomalaberrations,%Control 0.273 ± 0.024а 10.30 ± 0.35a 0.21 ± 0.05aJuice:water 1:10 0.164 ± 0.031b 1.25 ± 0.14b 0.18 ± 0.05aJuice:water 1:5 0.091 ± 0.013c 0.99 ± 0.13b ndModel solution:water 1:100.184 ± 0.027d 7.44 ± 0.32a 1.10 ± 0.13bModel solution:water 1:50.158 ± 0.010d 9.15 ± 0.32a 0.40 ± 0.07ba,b; a,c; a,d; c,d P ≤ 0.05; b,c P ≤ 0.1nd ‒ not detectedFigure 6 MDA concentration in onion roots treated with applejuice and model solutionscontroljuice 1:10juice 1:5juice 1:2020406080Growth in root mass,% of control**controljuice 1:20juice 1:10juice 1:5juice 1:202468Mitotic index, control fructose2 %020406080100Growth in root mass,% of controlcontrolfructose2 %fructose5 %fructose10 %fructose15 %-1012345678Mitotic index, %0510152025Control Juice 1:10 Juice 1:5 Modelsolution 1:10Modelsolution 1:5MDA concentration,μmol/g of fresh mass0510152025Control 1 %ethanol2 %ethanolJuice 1:2 Juice1:2+1%ethanolJuice1:2+2%ethanolMDA concentration,μmol/g of fresh massTable 3 Root mass growth, mitotic activity, and the frequencyof chromosomal aberrations of onion root meristem cells aftera day of treatment with apple juice (n = 5)Sample Growth inroot mass,g/onionMitoticindex, %Chromosomalaberrations,%Control 0.113 ± 0.018a 8.31 ± 0.31a 0.42 ± 0.07aJuice:water 1:10 0.099 ± 0.018a 4.57 ± 0.25b 0.17 ± 0.05bJuice:water 1:2 0.057 ± 0.011a 1.25 ± 0.12c 0.13 ± 0.04ba,b; a,c; b,c P ≤ 0.05326Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328Another observation we made was that adding 1 and2% ethanol to the juice did not increase the proliferativeactivity of the meristem cells. This result was quitepredictable since treating roots with ethanol solutionsdecreased the mitotic index of the meristem cells,compared to the control.CONCLUSIONOur study showed that apple juice manifestedsubchronic toxicity when it was diluted with water in aratio of 1:5 (~ 2% soluble solids). The toxicity caused a50% delay in the growth of Allium cepa roots, comparedto the control. At the same time, it sharply inhibitedthe division of meristem cells, with their mitotic indexdecreasing by a factor of 18 and the MDA concentrationincreasing by 11%. To identify the mechanisms ofthese disorders, we treated the roots with the maincompounds of juice dry solids – fructose, glucose,sucrose, D-sorbitol, and malic acid – and compared theabove indicators. We found that in contrast to the 1:5diluted juice, 2% fructose decreased the mitotic index byonly 17%, compared to the control. The model solutioncontaining 1.4% fructose, 0.4% glucose, 0.2% sucrose,0.1% D-sorbitol, and 0.06% malic acid showed a 40%higher growth in root mass compared to the 1:5 dilutedjuice (P < 0.05), the same mitotic index of meristemcells as the control, and a doubled concentration of MDAcompared to the control.Thus, the subchronic toxicity of apple juice primarilymanifested through its antiproliferative activity in themeristem cells. However, the above juice componentswere not involved in that activity. What they wereresponsible for was an increased level of lipid oxidationin the root tissues, which was restrained by the naturalantioxidants present in the juice.In addition, we analyzed the contribution of a foodadditive (ethanol) to the potential toxicity of apple juice,using the Allium test. We found that 1% ethanol in the1:2 diluted juice reduced the concentration of MDAin the roots by 30%, with no effect of acute toxicity inrelation to their growth.The above effects of, and relationships between,various biomarkers of apple juice and its componentscan form a basis for more detailed large-scale researchinto its safety. Our findings can also be used to studythe toxic potential of juice depending on manufacturingtechnology or food additives, as well as to create newjuice-based products.CONTRIBUTIONAll the authors were equally involved in writing themanuscript and are equally responsible for plagiarism.CONFLICT OF INTERESTThe authors declare that they have no conflict ofinterest.