In this study, it has been determined that 1,4-dioxane causes a versatile toxic effect in A.cepa meristemetic cells, which is a eukaryotic model organism, and t-resv reduces this toxicity. In 1,4-dioxane treated group significant reductions in radicle lenght, weight increase and germination rates were determined and high realtive injury rate was observed. Abnormalities observed in germination parameters may be related to toxic effects of 1,4-dioxane. Dioxane has direct or indirect toxic effects on living organisms. The direct effect occurs as a result of the reaction of 1,4-dioxane and molecular oxygen, which causes the production of free radicals and the formation of oxidative stress [13,14]. Oxidative stress in plants causes autocatalytic peroxidation of membrane lipids and pigments, modification of membrane permeability and consequently damage to cell structure [15]. In particular, the rapidly dividing meristem cells are more affected by oxidative stress, and root growth is inhibited and this inhibition is reflected in germination and weight gain. Similarly, it has been reported in the literature that application of 1,4-dioxane causes inhibition in weight gain, germination rate and root growth of test organisms [1,16]. T-resv showed an amerolative effect against 1,4-dioxane induced damage in germination parameters. Resveratrol is present in cis and trans isomeric form, and the concentration of the trans isomer, the main form, significantly contributes to biological activity [7]. In the literature, resveratrol is reported to be a potent antioxidant component that has been proven in various in vitro and in vivo studies. Chanbitayapongs et al. [11] reported that resveratrol inhibited metal-derived lipid peroxidation and showed antioxidant properties in in-vitro studies. Sun et al. [10] reported that resveratrol reduced lipid peroxidation induced by iron and ethanol. Since there is no data on the protective properties of t-resv against 1,4-dioxane toxicity in plants, this study is the first data on this subject.
MDA, GSH, SOD and CAT analyzes were performed to determine the effects of diosane and resve applications on antioxidant and oxidan balance. It was determined that dioxane application increased MDA level, decreased GSH level and induced antioxidant enzyme activities. It was found that these changes observed in antioxidant-oxidant balance started to normalize with resveratrol application. It is known that 1,4-dioxane causes free radical production and oxidative stress in living systems. Free radicals in the cells attack the unsaturated lipids containing carbon-carbon double bonds, causing lipid peroxidation. Low levels of MDA have been reported to act as regulators of gene expression in cells. However, high levels of MDA can easily interact with functional groups of molecules such as proteins, lipoproteins, DNA and RNA in the cell, causing adduct formation and different pathological conditions [17]. MDA is highly toxic and its toxicity is associated with Michael's ability to form adducts with thiol groups, facilitate protein cross-linking and induce mutagenesis [18]. Along with the increase in MDA, changes in glutathione levels were observed in meristematic cells treated with 1,4-dioxane. It was determined that glutathione level which is an antioxidant with tripeptide structure decreased 2.38 times in 1,4-dioxane treated group compared to control group. Briefly, the increase in MDA level and the decrease in GSH level in the same cells treated with 1,4-dioxane are indicative of obvious oxidative damage. Glutathions have been reported to be found in almost all cell parts of plant tissues such as cytosol, endoplasmic reticulum, vacuole, mitochondria, chloroplast, peroxisome. Reduced glutathione reacts with lipid peroxides and oxidizes during detoxification of these molecules and the level of reduced glutathione decreases [19]. In cells, the decreased glutathione level leads to reduced antioxidant capacity and the increased MDA level causes an enhanced oxidation. The decrease in GSH level and the increase in MDA level indicate the deterioration of antioxidant-oxidant balance. It was observed that this disrupted balance induced by 1,4-dioxane, started to improve with t-resv application. Resveratrol has scavenging activity on reactive oxygen species and shows significant effects on the radical induced cellular response [20]. Similarly, Al-Hussaini and Kilarkaje [21] reported that lipid peroxidation and macromolecule oxidation in the cell decreased with t-resv administration. In another study, Mikstacka et al. [22] reported that t-resv administration was effective in recovering significantly depleted GSH content in tested cells. Gupta et al. [23] reported that in oxidative stress-induced subjects the administration of 20 and 40 mg/kg t-resv administration resulted in a reduction in MDA levels, but no effect on GSH levels.
It has also been determined that 1,4-dioxane application induces SOD and CAT activities in A. cepa meristematic cells. SOD and CAT are involved in the removal of radical products in the cell. SOD catalyzes the dismutation of the highly reactive superoxide anion to O2 and the less reactive product H2O2 and the peroxide which is formed as a result of this reaction is destroyed by CAT enzyme [24]. Although there are studies showing that 1,4-dioxane causes oxidative damage and increases MDA level [25], there is no direct study investigating its effect on SOD and CAT activity. However, there are many studies in the literature that report changes in SOD and CAT activity in the presence of oxidative stress. Malar et al. [26] observed that in the presence of induced lipid peroxidation and oxidative stress SOD activity increased by 251% and CAT activity increased by 60% in Eichhornia crassipes leaf tissues compared to control. Macczak et al. [27] reported a reduction in GSH levels and alterations in SOD and CAT activities in the presence of oxidative stress caused by various chemicals inducing the formation of reactive oxygen species. In this study, t-resv treatment with dioxane ameliorated the enhanced SOD and CAT activity induced by 1,4-dioxane. These improvements can be explained by the role of t-resv in reducing oxidative stress and regulating enzyme induction. Similarly, Sadi et al. [28] specified that SOD activity increased in the presence of induced oxidative stress and decreased again after t-resv treatment, briefly they reported that t-resv treatment caused an improvement in the level of oxidative biomarkers. Pintea et al. [29] reported that t-resv directly contributes to antioxidant defense by scavenging reactive oxygen species and causing changes in superoxide dismutase, catalase and glutathione peroxidase activities.
1,4-dioxane, which causes oxidative stress in A. cepa meristematic cells, has also been found to cause chromosomal abnormalities such as fragment, sticky chromosome, unequal distribution of chromatine, bridge and vagrant chromosome. These genotoxic effects indicate that 1,4-dioxane disrupts genome stability, and these effects may be associated with 1,4-dioxane-induced oxidative damage. 1,4-dioxane leads to free radical formation, oxidative stress and lipid peroxidation in living systems [13,14]. The effects of oxidative stress on DNA have been investigated in detail and have been demonstrated by many studies in literature. Oxidative stress and free radicals cause DNA adducts, phosphodiester bond cleavages, chain breaks and mutations in bases [30]. Abnormalities in bases caused by 1,4-dioxane exposure cause A:T-T:A transversions and this mutation results from adenosine adducts [31,32]. All these changes in the DNA structure induced by 1,4-dioxane cause genome instability and the formation CAs. Similarly, Sağır et al. [16] reported that the application of 1,4-dioxane resulted in high CAs formations such as fragment, ascentric and dicentric chromosomes. In this study, it was also determined that genotoxic effects induced by 1,4-dioxane were decreased with t-resv treatment. Oxidative stress caused by 1,4-dioxane in organisms has been proved by the increased MDA levels in A. cepa root cells. Oxidative stress in cells also affects DNA and causes the formation of DNA base oxidation products. Agents that increase oxidative DNA damage increase cancer development. Inhibiting the formation of oxidative stress, which is the starting point of these abnormalities, will also inhibit mutation and cancer development. It has been shown in many studies that consumption of antioxidant-containing plants in daily diet reduces oxidative DNA damage levels and the incidence of human cancers [33]. Resveratrol increases the expression of glutathione peroxidase and catalase enzymes and induces scavenging of H2O2, thereby providing resistance to oxidative damage [34]. Agents such as resveratrol that reduce oxidative stress and consequently DNA damage have antimutagenic and anti-cancer effect. Jang et al. [35] reported that resveratrol has an anticarcinogenic effect and this effect is associated with inhibition of the initiation and promotion of carcinogenesis. Al-Hussaini and Kilarkaje [21] reported that DNA oxidation in the cell decreased in subjects treated with t-resv. Ungvari et al. [34] showed that DNA damage induced by oxidative stressor was reduced by 10-6–10-4 mol/L resveratrol application in a cell culture.
The cytogenetic effects of 1,4-dioxane and t-resv applications in meristematic cells were also supported by MN and MI analysis. It has been proven by the increase in the frequency of MN and the decrease in MI rate that 1,4-dioxane causes genomic instability. The decrease in the MI ratio of 1,4-dioxane treated group can be explained by the oxidative stress created in the meristematic cell. Chemicals that trigger the formation of oxidative damage and cause glutathione reduction are reported to delay cell cycle by causing delayed progression through G1 and S phases and even stopping the cell cycle at the G2 point [36]. The fact that the 1,4-dioxane application causes an increase in MDA level, a decrease in gsh level and changes in antioxidant enzyme activities in meristematic cells is evidence of oxidative damage and this damage causes an inevitable delay in cell cycle. Similarly, Sağır et al. [16] reported that 1,4-dioxane administration caused a decrease in the MI rate and reduced the number of dividing cells from 835±45.38 to 438.5±23.31. The decrease in MI ratio also explains the abnormalities caused by 1,4-dioxane in physiological parameters. Root growth, germination and weight gain are directly related to cell division, and the decrease in division rate negatively affects these parameters. DNA damage and MN formation also cause disruptions and abnormalities in the cell cycle. The formation of MN in a cell is an indicator of toxic effect and MN is caused by all chromosomes or chromosome fragments that do not belong to the main nucleus. MN is usually caused by abnormalities in the mitotic spindle, kinetocor or mitotic apparatus and chromosomal damage [37,38]. The fact that MN induction in meristematic cells indicates that 1,4-dioxane causes mitotic abnormalities and has genotoxic effect. Similarly, Teker et al. [1] reported that 1,4-dioxane treatment caused an increase in MN frequency in root tip cells in a dose dependent manner and they achieved a MN level of 46.70±11.91 in the group treated with 100 ppm 1,4-dioxane. In this study, the protective properties of t-resv proven in previous parameters were also observed in MN and MI analysis. T-resv application with 1,4-dioxane was found to cause improvement in MN and MI rates. This protective property of t-resv can be explained by its reducing effect against oxidative damage induced by 1,4-dioxane. In the previous analysis of this study, t-resv was found to reduce the rate of increased MDA and regulate the GSH level, thereby reducing oxidative damage. These results are the evidence that t-resv administration is protective against oxidative stress and same amerolative effect was also observed in MN and MI results. Similarly, Ranjini and Manonmani [39] reported that 100 µM resveratrol treatment has protective effects against induced toxicity and has a reducing effect on MN formation and an an enhancing effect on cell viability.
The application of 1,4-dioxane has been found to cause changes such as flattened cell nuclei, cell wall thickening and cell deformation in the anatomical structure of A.cepa root. Root tissue can develop various adaptation mechanisms against toxic agents. Thickening of the cortex cell wall is one of the adaptation mechanisms, thereby reducing the access of the toxic agent to the internal tissues and the central cylinder [40,41]. In addition to the general structure of the cell, anatomical changes were observed in the cell nucleus. Flattening of the cell nucleus was observed with 1,4-dioxane application and this was associated with the cumulative effect of genotoxic and biochemical changes caused by 1,4-dioxane. A possible change in intracellular pressure and cell skeleton as a result of toxicity that disrupts the overall integrity of the cell may lead to shape changes of the organelles. The cell nucleus is generally spherical or ellipsoidal, but may change its shape in response to intracellular changes. Alterations in nuclear volume and protein concentration, degradation in DNA integrity and density may result in nuclear shape changes [42,43]. 1,4-dioxane induced peroxidation in membrane component lipids, disruption of DNA integrity by MN and CAs formations are the possible explanation for nuclear shape changes in Group IV. Although there is no study on the effect of 1,4-dioxane on anatomical damage, many studies have shown that many toxic agents cause changes and necrosis in epidermis and cortex cells [44,45]. With previous analyzes of this study it was found that 1,4-dioxane induced genotoxic effects and abnormalities in antioxidant system were found to be reduced by t-resv. Ameliorative effects of t-resv in other parameters have also been shown against anatomical damages. treatment with t-resv caused a significant decrease in anatomical changes especially in the frequency of flattened cell nucleus. Similar, Macar et al. [45] reported that 400 mg/L and 800 mg/L resveratrol application alleviate the anatomic damages in meristematic cells induced by CuCl2.