​Antioxidant Responses of Arsenite-induced Oxidative Stress in Rice (Oryza sativa L.) and its Modulation by Eugenol (Extracted from Ocimum sanctum)

Arsenic, one of the most dangerous toxicants to the global environment is causing serious health problems in South East Asia especially in West Bengal and Bangladesh with elevated concentrations of up to 3200 µg/L in drinking water. (Carty et al., 2011) Arsenic-contaminated water irrigation of soils significantly increases the concentrations of arsenic in the soil, which adversely affects various physiological and biochemical abnormalities (Li et al., 2006) in plants and humans. In humans, arsenic toxicity is associated with several chronic diseases that involve skin, bladder, lung, liver and kidney cancer (Mandal et al., 2002; Waalkes et al., 2004; Mehmood et al., 2017). While in plants, arsenic uptake adversely affects the metabolic processes and causes several physiological and cellular disorders and hinders the growth and development in various manners (Singh et al., 2015). Arsenic induces oxidative stress due to excessive production of reactive oxygen species causing damage to the cell membranes, lipids, proteins and eventually cell death (Srivastava et al., 2007) by activating oxidative signaling pathways. Against such oxidative stress, plants develop an integrative defense system including both enzymatic and non-enzymatic antioxidants. These reactive oxygen species can be controlled by increasing the production of various enzymes such as APX, GPX, SOD, CAT, glutathione reductase and externally supplied antioxidants, especially polyphenolic compounds. Eugenol is a phenylpropanoid phenol that can serve as an antioxidant or pro-oxidant agent. It also has cytotoxic, anti-carcinogenic and anti-mutagenic effects (Bezerra et al., 2017) and is used to suppress the activation of nuclear factor kappa B (NF-kβ) in mouse skin with TPA-induced inflammation (Kaur et al., 2010). Rice is also one of the important crops in the arsenic-contaminated region and rice arsenic toxicity has newly been subjected to immense adversity (Mehrag et al., 2004). No studies were reported at current on the role of exogenous eugenol treatment in arsenic-induced oxidative damage and antioxidant defense systems in rice seedlings to the best of knowledge.
       
The present study examined the role of treatment of rice seeds with herbal extract eugenol to modulate the oxidative stress in Oryza sativa L., induced by arsenic toxicity.

Chemical and reagent
 
Analytical grade chemicals were used for the research and purchased from Sigma-Aldrich. From the leaf the Ocimum sanctum plant, eugenol was extracted and characterized by element detection, IR, NMR and other parameters.
 
Extraction, isolation and identification of eugenol from Ocimum sanctum (Tulsi)
 
Eugenol is extracted from the essential oil of tulsi leaves by co-distillation with indirect steam and separated from the distillate by using an organic solvent such as dichloromethane. Finally isolated by evaporation of the organic solvent and dried by anhydrous sodium sulfate desiccator, b.p. 248°C.
       
Eugenol was characterized by elemental analysis, IR, NMR spectra.
 
Anal (C₁₀H₁₂O₂)
Found                          : C 73.28; H 7.26; O 19.46
Required                      : C 73.14; H 7.37; O 19.49
IR (KBr)                        : 3516(OH), 1268, 1235 (C-OCH₃),
                                       1638 (C=C, alkene), 1613, 1516
                                       (aromatic (C=C) and 820 (Benzene ring substitution) cm^-1
NMR (CDCl3) (ppm)     : 4.36 (s, 3H,-CH₃), 3.73 (s, 3H, -OCH₃),
                                       6.82 (d,2H, ArH), 6.72 (s,1H, ArH),
                                       6.38 (bh, 1H, -OH), 5.18 (m, 2H, CH₂
                                       =CH), 6.02 (M,1 H, CH₂=CH-CH₂)
 
Hydroponic medium and treatments
 
Oryza sativa L. seeds of specific genotype (IR 64) arranged from IARI, New Delhi were screened in hydroponic condition  (Daveet_al2013) for antioxidant enzymes and oxidative stress markers assay in shoot and root respectively during arsenite and eugenol exposure. For the experimental procedure, seeds were sterilized with 0.1 per cent solution of HgCl₂ for about 0.5 minutes, after seeds were washed 3-4 times by distilled H₂O and soaked in double-distilled water for one day. These seeds were then transferred to Petri-dishes and kept in the culture room at 27°C in dark for proper germination for up to seven days. After that these seedlings were exposed to arsenite, eugenol separately and co-treatment (10, 50 and 100 µM) for 7 days under similar conditions. All treatments were in triplicate. After seven-day treatment, plants were harvested, rinsed by double-distilled water and used for different parameters.
 
Antioxidant enzymes and oxidative stress markers assay
 
Treated and untreated rice seedling samples were homogenized in a chilled mortal with 3 ml 0.1M Na₃PO₄ buffer at seven pH having 1.0% polyvinyl polypyrrolidone, 1.0 mM disodium-EDTA and 0.5 M sodium chloride. The amalgamate were centrifuged at 10,000 rpm about 15 minute at 4°C and the supernatant was utilized to determine APX, SOD, CAT, GPX activities. The ascorbate peroxidase was estimated according to (Nakano and Asada 1987) by assessing the ascorbate oxidation rate. The superoxide dismutase exploit was measured as per (Beauchamp - Fridovich 1971) via assessing the inhibition of the reduction of NBT-dye by superoxide dismutase anion. The GPX-activity was estimated as per (Hammer Schmidt et al., 1982). The catalase action was assessed according to (Aebi et al., 1983). The malondialdehyde content was assessed as per the modified process of (Hodges et al., 1999).
 
Statistical analysis
 
All investigations in triplicate were statically analyzed by analysis of variance (ANOVA) to confirm the variability of data and validity of results and Duncan’s multiple range test (DMRT) was performed to determine the significant differences between treatments at 0.05.

The 7-day old eugenol treated seedlings were analyzed for different parameters: antioxidant activity (APX, CAT, SOD, GPX), lipid peroxidation [malondialdehyde (MDA) content], exposed to applied concentrations (100 µM, 50 µM and 10 µM) of arsenite, eugenol and their joint exposure were shown in Fig 1, 2.
 

 

 
Malondialdehyde (MDA)
 
The MDA increased by 100%, 76%, 26% in root while by 90%, 70%, 22% in the shoot at 7 d treatment by 100 µM, 50 µM,10 µM AsIII respectively as compared to control (R=0.964221, P=0.05). Upon eugenol treatment under the same days and same concentration as AsIII treatment, the MDA decreased only by 16%, 8%, 4.25% in root while in shoot 19%, 8.3%, 3.5% as compared to control. Upon joint application of eugenol with AsIII, the increased levels of MDA in root was reduced by about 46%, 36%, 16% while in shoot 40%, 35% and 14% as compared to AsIII treated rice seedling under the same concentration only and was positively correlated with As accumulation (R=0.94617, P=0.05) as shown in Fig 1.
 
Effect of antioxidant
 
In comparison with control, the APX activity increased by 78%, 37% and 30% in the root, while by 77%, 36%, 28% in the shoot at 7 d treatment with 100 μM, 50 μM, 10 μM As III (R=0.937097, P=0.05). The activity of APX decreased by 26%, 21%, 19%, in the root, while in shoot 38%, 19%,15%  on eugenol treatment under the same day and same
concentration as AsIII treatment as compared to control. Upon joint exposure of eugenol with AsIII, the increased root APX level was reduced by approximately 61%, 30%, 24%, while the shoot was by approximately 64%, 28%, 13% compared to AsIII treated rice seedling under the same concentration only (R=0.859663, P=0.05) (Fig 2A).
       
The CAT activity increased by 34%, 28%, 14% in root while by 29%, 23%, 12% in shoot at 7 d treatment with 100 µM, 50 µM, 10 µM AsIII respectively (R=0.93181, P=0.05), while its level decreased by 20%, 11%, 7.2% in root and in shoot by 13%, 8%, 5.3% as compared to control under the same conditions as arsenite treatment. Eugenol when jointly treated with AsIII, the increased level of CAT in root was reduced by about 19%, 13%, 6% while in shoot 18%, 11%, 6% as compared to only AsIII treated rice seedling under the same concentration only (R=0.94617, P=0.05) (Fig 2B). GPX activity increased by 41%, 29%, 21% in the root, whereas by 34%, 26%, 15% in the shoot with 100 μM, 50 μM, 10 μM AsIII at 7 d treatment (R=0.918705, P=0.05) while its value decreased by 17%, 5%, 1.8% in root whereas in shoot 12%, 4.6%, 1.5% with 100 μM, 50 μM, 10 μM eugenol treatment as compared to control. The root GPX activity decreased on average by about 29%, 15%, 12% while in shoots 15%, 12%, 7.6%, with the joint application of eugenol with AsIII, compared with only AsIII treated rice seedling with the same concentration only (R=0.935489, P=0.05) (Fig 2C). The SOD activity in the root improved by 34%, 28%, 14%, while shoot 29%, 23%, 12% with 100 μM, 50 μM, 10 μM As III at 7d treatment compared to control (R=0.953166, P=0.05). The SOD declined by 27%, 24%, 15% in root while in shoot 21%, 17%, 8% during eugenol treatment under the same condition as AsIII treatment, compared to control. The increased level of SOD in the root was decreased by approximately 46-53% whereas in shoots 31-35% upon joint application of eugenol with AsIII treated rice seedling under the same concentration as As III treated only and was positively associated with As accumulation (R=0.99218, P=0.05) (Fig 2D).
       
Treatment of rice (O. sativa) seedling with eugenol, when investigated with arsenite treatment in hydroponic, showed a significant result for MDA, antioxidant enzymes as compared to only found by arsenic-treated rice seedling alone.
       
Substantial augmentation of APX activity on arsenic exposure can rely on greater availability of H₂O₂ due to the effective breakdown of the ASC-GSH cycle, whereas substantial reduction of the APX value when treated with eugenol may be attributable to the neutralizing adverse effects of arsenic due to complex formation and a reduction of O₂^- to molecular oxygen (Gautam et al., 2019) resulting in decreased H₂O₂ levels and supported by (Dave et al., 2013; Mairaj et al., 2020; Souri et al., 2017 ). The activity of the CAT, GPX enzyme in the root and shoot was reduced on average when exposed to eugenol in arsenite treated rice seedlings, suggesting that eugenol causes a significant reduction in oxidative stress caused by toxic arsenic and well supported by (Gautam et al., 2019). Findings also suggest that during arsenic stress, enhanced SOD activity may be correlated with H₂O₂ production. This arsenic stress has been ameliorated by lowering the levels of H₂O₂ when treated with eugenol. In this way, eugenol plays a protective role against oxidative stress caused by arsenic. High dose exposure of eugenol in a hydroponic medium decreases the activity of the enzymes in rice plants exposed to arsenic and helpful in reducing arsenic-mediated rice plant toxicity (Gautam et al., 2019). In the present study, it has been noted that the MDA level increased with arsenic exposure in rice seedlings, which may be attributed to the excessive production of reactive oxygen species such as O₂^-, OH, H₂O₂ under stressed condition by the interaction of arsenic with intracellular components (Dave et al., 2013; Chandrakar et al., 2017b). The similar results regarding the up-regulation of MDA activity were also noted in arsenic exposed Zea mays L., Withaniasomnifera, Oryzatenuiflorum L., Glycine max L. and supported by (Hartley- Whitaker et al., 2001; Srivastava et al., 2005; Anjum et al., 2016).

Taking into account the findings, arsenic exposure in rice plants is presumed to adversely affect the antioxidant defense system by excessive production of reactive oxygen species causing oxidative stress in terms of enhanced H2O2 and may increase the activity of antioxidative enzymes and oxidative stress marker which was correlated with arsenic accumulation. Arsenite supplementation with eugenol induces a substantial reduction in the activity of APX, CAT, GPX, SOD and MDA levels in the root and shoot of the rice plant. This study suggests that arsenic-induced oxidative stress in rice seedlings has been substantially improved by treatment with eugenol.

Amity University, Uttar Pradesh is highly acknowledged for providing the infrastructure and facilities for the work done.