Synergistic effects of selenium and silicon mitigate arsenic toxicity in plant (Oryza sativa L.) and human model (keratinocyte HaCaT cells lines) systems

doi:10.21203/rs.3.rs-2130617/v1

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Synergistic effects of selenium and silicon mitigate arsenic toxicity in plant (Oryza sativa L.) and human model (keratinocyte HaCaT cells lines) systems

https://doi.org/10.21203/rs.3.rs-2130617/v1

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Arsenic (As) is a toxic element for plants, animals and humans. The mitigation of As stress has been achieved via selenium (Se) and silica (Si) supplementations. However, the effects of combined application of Se and Si against As stress are not known. The present study was conducted on a plant (rice; Oryza sativa L.) and human model (keratinocyte HaCaT cell lines) systems to evaluate the effects of Se (0.5 and 1 mg L^− 1) and Si (10 and 30 mg L^− 1) against As (4 mg L^− 1). The rice plants, grown hydroponically for 7 d in various treatments, showed reduction in As accumulation in As + Se/Si/Se + Si treatments in comparison to As treated plants. The reduction in As accumulation in root and shoot was 57% and 64%, respectively in As + Se + Si treatment. The increase in enzymatic antioxidant system (SOD, APX, GPx, GR and GST) along with decrease in oxidative stress markers (MDA, DHA, ASC and H₂O₂) in As + Se + Si treatment as compared to As treatment signified elevated tolerance of rice plants to As stress. The cytotoxicity amelioration by Se + Si supply was also seen in HaCaT cells exposed to As in terms of cell viability and As-induced shrinking and apoptosis. In conclusion, the results demonstrated that an optimum combination of Se and Si can be effectively used to mitigate As toxicity in plants and possibly humans also.

Antioxidants

Arsenic

HaCaT cell lines

Rice

Selenium

Silica

Rice is the staple food in South and Southeast Asian countries and consumed by 50% of the world population (Khush 2013; Srivastava et al. 2022). Urbanization, industrialization, and continuous population growth not only enhances the food demand but also forces to meet this demand on dwindling natural resources. Agriculture practices, such as intensive over cropping and use of short-duration varieties are in practice to meet the demand for rice. However, these agricultural practices negatively affect the nutrient quality of rice grain. This may result in a deficiency of nutrient elements in people. Besides mineral deficiency, augmentation of the toxic element viz., arsenic (As), chromium (Cr), lead (Pd), cadmium (Cd) etc. in the food system induces the development of chronic diseases like skin diseases, lung infection, and cancers in human being (Jaishankar et al. 2014; Chakraborty et al. 2022). Being a non-essential and toxic element, As is known to decrease the nutrient quality and yield of rice crops (Cooper et al. 2020; Upadhyay et al. 2022). Arsenic competes with structurally analogous essential minerals for entry into the plants (Awasthi et al. 2017; Kumar et al. 2020a) and this has been the basic stimulant for research on As-elemental interactions. Several elements like selenium (Se), silica (Si), sulphur (S), phosphorus (P), and zinc (Zn) are being used to improve plant growth, yield and productivity and for amelioration of As induced toxicity (Kumar et al. 2014b, 2016; Dixit et al. 2015; Srivastava et al. 2016; Sahay et al. 2020; Yang et al. 2020; Dwivedi et al. 2020).

Selenium (Se) is well known trace element that counteracts toxic effects of As in the plants (Kumar et al. 2016, Moulick et al. 2016; Pandey and Gupta 2018; Bhadwal and Sharma 2022; Das et al. 2022) due to homology to As along with similar physical and chemical properties (Rosen et al. 2009). Selenium is taken up in the form of SeO₂^3- by the rice and acts as a co-factor of different –SH containing compounds assisting in detoxification process. Recent studies demonstrated that Se minimized As uptake and translocation in rice by competitively interacting with As (Kumar et al. 2016; Bhadwal and Sharma 2022). Silica (Si) is 2^nd most abundant element in earth crust. It is beneficial to growth and development of rice crops and has been reported to provide tolerance against various abiotic stresses (Coskun et al. 2016; Dwivedi et al. 2020). Silicon is a vital component for growth and yield of rice and assists in restraining the entry of arsenite (AsIII) to the plant through silicic acid pathways (Gao et al. 2021). Dwivedi et al. (2020) reported that orthosilicic acids reduced the rice grain As accumulation and enhanced the plant growth.

Arsenic contamination in rice is a major source of inorganic As to human population dependent on rice as a staple food. In human beings, As poisoning leads to skin cancer, bladder cancer, diminished nerve conduction, irritability, anxiety, memory loss and headache inability (Mitra et al. 2020). Therefore, assessment of As poisoning and its cure are necessary and to this end, cell lines act as a potential tool to perform cytotoxicity studies in vitro (Banerjee et al. 2022). HaCaT cell lines are the most common type of cell lines used to assess the effect of toxins and other metabolites at morphological and molecular level (Ammerman et al. 2009) in humans. These are the immortalized human keratinocytes, which have been utilized as an experimental model to examine the epidermal homeostasis and its pathophysiology. HaCaT cells are very close to tumorigenesis (Weinmuellner et al. 2018).

In present study, cytotoxic assessment of As has been done in rice, and cell line model to characterize the level of toxicity and to evaluate the protective role of Se and Si alone and in combination. The hypothesis was that competitive nature of Se and Si against As would reduce As accumulation in plant / human model systems and would be helpful in reducing As toxicity.

Growth condition and experimental design

The healthy rice seeds (Sarjoo-52) were obtained from National Seed Corporation Limited, Lucknow, India. The seed were surface sterilized using 0.01% hydrogen peroxide for 45 s and washed with double distilled water. Seeds were germinated in moist pre-sterilized blotting sheets on a tray in seed germinator for 4 d at 25 ^oC at 65% relative humidity. The 4 d old seedlings were transferred to perforated cups with 10 seedlings / cup. The cups were placed in trays holding 3 L of full-strength Hewitt nutrient medium prepared in Milli-Q water (Liu et al. 2004). The seedlings were grown for 10 d under 210 µmol cm^− 2 s^− 1 (16/8 h day/night) white florescent light in a culture room maintained at 24–26 ^oC and then exposed to different concentrations of As (4 mg L^− 1; using sodium arsenite, Na₂AsO₂), Se (0.5 and 1 mg L^− 1; using sodium selenite, Na₂SeO₃) and Si (10 and 30 mg L^− 1; using sodium silicate, Na₂SiO₃) for 7 d. All the experiments were performed in four replicates in controlled light, temperature and humidity in culture room. Plants were harvested, washed with milli-Q, separated into roots and shoots, blotted and used for the study of various parameters. All the samples were ground in liquid N₂ and stored at-80 ^oC till further use.

Plant Growth Measurement And Photosynthetic Pigments

Root and shoot lengths were measured by metric scale. Total fresh weight of As exposed and control plants was also noted. Chlorophyll content of treated and untreated plants (100 mg) were assayed according to Arnon (1949) while carotenoid content was estimated as per Duxbury and Yentsch (1956).

Quantification Of Arsenic, Selenium, Silica And Quality Control

The level of As and Se were determined following Kumar et al. (2013). Plant tissues (500 mg) were digested using HNO₃. Digested sample were filtered and volume was made up to 10 ml using Milli-Q water. The As and Se were analyzed using AAS (GBC AvantaS). Silicon content was determined following the method of Vender Vorm (1987) and Dwivedi et al. (2020). The whole experiment was set up in the randomized block design. Analytical data quality of the elements was ensured through repeated analysis (n = 3) of standard reference material. Certified reference material (CRM 028–050) used for the accuracy of the AAS was soil sample procured from Resource Technology Corporation, USA (Lot no. IH 028).

Specific Metal Accumulation And Translocation Factor

Translocation factor (TF) for As was calculated as per the following formula:

TF = shoot / root (As concentration).

Specific As uptake (SAU), specific Se uptake (SSeU) and specific Si uptake (SSiU) were calculated according to Zhang and Duan (2008) with slight modification as reported in Dwivedi et al. (2010).

Estimation Of Superoxide Radical (O), Thiobarbituric Acid Reactive Substances Hydrogen Peroxide, And Electrical Conductivity

The level of superoxide radical (O₂^•−) production was measured following the

method of Chaitanya and Naithani (1994). Lipid peroxidation (MDA) and Hydrogen peroxide (H₂O₂) content were estimated according to the method of Heath and Packer (1968) and Velikova et al. (2000) respectively. The ion leakage was measured in terms of electrical conductivity (EC) according to Devi and Prasad (1998).

Estimation Of Oxidative Stress And Activity Of Antioxidant Enzymes

For the analysis of enzyme activities, fresh shoot samples (500 mg each) were prepared by homogenizing in buffer (pH 7.5) specific for each enzyme. The homogenate was centrifuged at 12,000× g for 15 min at 4 ⁰C. All the steps in the preparation of enzyme extract were carried out at 4 ⁰C. The proteins of the shoot samples were extracted following particular enzyme activity methods. The quantification of protein in plant samples was performed following the Bradford (1976) method. The superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), guaicol peroxidase (GPX, EC 1.11.1.7), dehydroascorbate reductase (DHAR), ascorbate oxidase (AOX, EC 1.10.3.3), glutathione peroxidase (GPx, EC 1.11.1.9), glutathione reductase (GR) and glutathione S-transferase (GST, EC 2.5.1.18) activities were measured following the method of Rai et al. (2011), Kumar et al. (2014b) and Dixit et al. (2016) respectively. Assay of cysteine synthase (CS; EC 2.5.1.47), γ-gluta-mylcysteine synthetase (γ-ECS; EC 6.3.2.2), and serine acetyl transferase (SAT, EC 2.3.1.30) activities was performed following Saito et al. (1994) and Seelig and Meister (1984), respectively, with slight modifications (Kumar et al. 2014a, b).

Cytotoxicity Assay In Hacat Cells

For evaluation of effects of elemental combinations on the cytotoxicity of As, humans HaCaT cell line were used. HaCaT cells were cultured and maintained in RPMI (Sigma) supplemented with 10% FBS (Life Technologies) and antibiotic-antimycotic solution (Sigma) in presence of 5% CO₂ at 37°C. Trypsinized 5000 cells /well were seeded in 96 well plate and incubated overnight. Then, 100 µL fresh media (MEM) was added to each well and treated with different concentrations (mg L^− 1) of As, Se and Si [As (4), Se (0.5), Si (30), As(4) + Se (0.5), As (4) + Si (30) and As (4) + Se (0.5) + Si (30)] were added in triplicates. Plate was incubated for 72 h at 37°C in 5% CO₂. After incubation, 10 µl/ well of viability marker dye Resazurin (stock solution 12.5 mg/100 ml PBS) was added and the plate was incubated for another 24 h. Fluorimetric reaction was measured using an excitation wavelength of 536 nm and an emission of 588 nm in a fluorimetric plate reader (BMG Labtech).

Cytomorphological Analysis By Phase-contrast Microscopy

Morphological variations were observed after exposing the HaCaT cells (human keratinocyte cell line) with different combination (mgL^− 1) of As, Se and Si [As(4), Se(0.5), Si(30), As(4) + Se (0.5), As(4) + Si(30) and As(4) + Se(0.5) + Si(30)]. An inverted phase-contrast microscope (Labomed, U.S.A) was used to observe the changes in the morphology of the treated cells in comparison with that of the untreated cells (control).

Statistical analysis

All values are average of 4 replicates. Duncan’s multiple range test (DMRT) was performed to determine the significant difference between treatments by using SPSS 21.0 software. Correlations among the parameters were calculated and drawn using Microsoft Excel- 2016.

Impact on growth parameter and photosynthetic pigments

Growth parameters under control and different treatments of As, Se and Si are presented in Table 1. Arsenic treatment to rice plants inhibited the root length (24%), shoot length (34%) and seedling biomass (27%). Arsenic influenced the root growth by altering root thickness, length, and biomass with different Se and Si alone and combination treatments (Fig. S1). Treatments of rice plants with Se and Si exhibited marked increase in growth characteristics. Silica treated rice plants exhibited better response as evidenced by increased root and shoot length and biomass in comparison to Se alone. However, co-application of Se + Si to As stressed rice plants showed the maximum increase in root length (81%), shoot length (75%) and biomass (69%) as compared to As alone treated plants. The As exposure significantly negatively altered the photosynthetic pigments (Fig. S2 A-D); the effect was more pronounced on the chlorophyll a (32%) than chlorophyll b (23%). Photosynthetic pigments demonstrated increase in response to Se and Si supplementation with the maximum increase being in combined Se + Si supplementation as compared As alone treatment.

Table 1

Effect of different metal(loid) supplementation on roots, shoots length and biomass of rice plant during arsenic stress.
Treatments (mg kg^− 1)	Root length (cm)	Shoot length (cm)	Biomass (g fw)
Control	4.27 ± 0.29^b	11.25 ± 1.30^bcde	2.99 ± 0.19^bcde
As (4)	3.24 ± 0.21^a	7.47 ± 0.58^a	2.18 ± 0.12^a
Se (0.5)	4.43 ± 0.27^bc	13.17 ± 1.15^ef	3.23 ± 0.20^cdefg
Se (1)	4.65 ± 0.48^bcd	13.89 ± 0.82^f	3.37 ± 0.19^efgh
Si (10)	4.58 ± 0.39^bcd	13.65 ± 1.46^f	3.26 ± 0.23^defg
Si (30)	5.135 ± 0.62^cdef	14.27 ± 1.04^f	3.61 ± 0.22^gh
As (4) + Se (0.5)	4.28 ± 0.48^b	10.65 ± 1.09^b	2.78 ± 0.28^b
As (4) + Se (1)	4.22 ± 0.58^b	10.97 ± 1.41^bc	2.90 ± 0.24^bcd
As (4) + Si (10)	4.19 ± 0.36^b	10.59 ± 0.86^b	2.83 ± 0.17^bc
As (4) + Si (30)	4.34 ± 0.46^bc	11.14 ± 1.07^bcd	3.06 ± 0.18^bcde
As (4) + Se (0.5) + Si (10)	4.78 ± 0.44^bcde	12.48 ± 0.95^bcdef	3.12 ± 0.18^bcdef
As (4) + Se (1) + Si (10)	5.29 ± 0.38^def	12.67 ± 0.81^cdef	3.25 ± 0.25^defg
As (4) + Se (0.5) + Si (30)	5.87 ± 0.40^f	13.04 ± 0.86^def	3.69 ± 0.20^h
As (4) + Se (1) + Si (30)	5.48 ± 0.61^ef	12.83 ± 0.97^cdef	3.48 ± 0.28^fgh
All the values are means of 3 replicate (n = 3) ± S.D. ANOVA significant at p ≤ 0.01. Different letters indicate significantly different values between treatments (DMRT, p ≤ 0.05).

Arsenic, Selenium And Silica Accumulation

The maximum accumulation of As, Se, and Si in single treatments was 376 µg g^− 1 dw, 15 µg g^− 1 dw and 87 mg g^− 1 dw, respectively in root and 114 µg g^− 1 dw, 10 µg g^− 1 dw and 35 mg g^− 1 dw, respectively in shoot. The As accumulation was significantly decreased in As + Se and As + Si exposed rice plants. However, plants exposed to As with combination of Se + Si showed the maximum decrease in As accumulation in root (57%) and shoot (70%) in comparison to As alone treatment (Table 2). Specific arsenic uptake (SAU) was the maximum in As alone treatment (105 µg g^− 1 dw). The TF of As was 0.303 ± 0.023 in As alone treatment; however, supplementation of Se + Si with As led to decrease in TF to 0.211 ± 0.019. The TF showed the maximum transfer of Se and Si to be 0.650 and 0.405, respectively (Table 3).

Table 2

Effect of different metal(loid) supplementation on uptake of As, Se and Si concentration in root and shoot of rice plant under As treatments.
Treatments (mg kg^− 1)	As (µg g^− 1 dw)		Se (µg g^− 1 dw)		Si (mg g^− 1 dw)
Treatments (mg kg^− 1)	Shoot	Root	Shoot	Root	Shoot	Root
Control	-	-	-	-	-	-
As (4)	114 ± 2.15^g	376 ± 32.88^e	-	-	-	-
Se (0.5)	-	-	4.45 ± 0.41^b	9.88 ± 0.33^a	-	-
Se (1)	-	-	9.83 ± 0.52^d	15.11 ± 0.44^b	-	-
Si (10)	-	-	-	-	19.87 ± 2.40^ab	55.95 ± 7.28^ab
Si (30)	-	-	-	-	35.29 ± 4.34^d	87.16 ± 10.07^d
As (4) + Se (0.5)	70.07 ± 3.55^ef	272 ± 23.77^cd	3.56 ± 0.26^a	10.46 ± 0.11^a	-	-
As (4) + Se (1)	65.20 ± 5.72^de	287 ± 23.08^d	7.95 ± 0.33^c	14.04 ± 1.40^b	-	-
As (4) + Si (10)	77.66 ± 7.44^f	302 ± 26.44^d	-	-	16.32 ± 2.30^a	42.72 ± 7.93^a
As (4) + Si (30)	61.51 ± 5.21^cd	273 ± 26.20^cd	-	-	29.85 ± 3.18^c	83.33 ± 8.86^d
As (4) + Se (0.5) + Si (10)	53.65 ± 4.69^bc	223 ± 19.51^b	3.52 ± 0.38^a	10.98 ± 0.96^a	23.46 ± 2.05^b	73.73 ± 7.07^cd
As (4) + Se (1) + Si (10)	50.66 ± 4.08^b	235 ± 22.48^bc	8.45 ± 0.68^c	23.56 ± 2.14^c	20.56 ± 1.65^ab	62.28 ± 4.57^bc
As (4) + Se (0.5) + Si (30)	33.98 ± 3.83^a	161 ± 14.04^a	4.14 ± 0.34^ab	11.13 ± 1.07^a	30.54 ± 3.28^cd	81.45 ± 9.15^d
As (4) + Se (1) + Si (30)	47.78 ± 4.18^b	177 ± 14.27^a	8.23 ± 0.63^c	22.38 ± 1.61^c	32.78 ± 2.72^cd	86.16 ± 9.29^d
All the values are means of 3 replicate (n = 3) ± S.D. ANOVA significant at p ≤ 0.01. Different letters indicate significantly different values between treatments (DMRT, p ≤ 0.05).

Table 3

Transfer factor (TF) and Specific arsenic uptake (SAU) in rice plant under different metal(loid) treatments.
Treatments (mg kg^− 1)	TF (As)	TF (Se)	TF (Se)	SAU
Control	-			-
As (4)	0.303 ± 0.023^d			105.48 ± 9.98^d
Se (0.5)		0.450 ± 0.039^b		-
Se (1)		0.650 ± 0.049^d		-
Si (10)			0.355 ± 0.030^abc	-
Si (30)			0.405 ± 0.031^c	-
As (4) + Se (0.5)	0.258 ± 0.021^bc	0.340 ± 0.039^a		50.84 ± 4.05^c
As (4) + Se (1)	0.227 ± 0.020^ab	0.566 ± 0.046^c		49.40 ± 3.80^c
As (4) + Si (10)	0.257 ± 0.024^bc		0.382 ± 0.030^bc	57.91 ± 4.96^c
As (4) + Si (30)	0.225 ± 0.018^ab		0.358 ± 0.034a^bc	49.60 ± 4.72^c
As (4) + Se (0.5) + Si (10)	0.241 ± 0.025^abc	0.320 ± 0.026^a	0.318 ± 0.024^a	38.11 ± 2.89^b
As (4) + Se (1) + Si (10)	0.216 ± 0.018^a	0.359 ± 0.031^a	0.330 ± 0.031^ab	36.56 ± 3.46^b
As (4) + Se (0.5) + Si (30)	0.211 ± 0.019^a	0.372 ± 0.028^a	0.375 ± 0.028b^c	24.18 ± 2.01^a
As (4) + Se (1) + Si (30)	0.269 ± 0.017^cd	0.368 ± 0.023^a	0.380 ± 0.030b^c	29.98 ± 2.39^ab
All the values are means of 3 replicate (n = 3) ± S.D. ANOVA significant at p ≤ 0.01. Different letters indicate significantly different values between treatments (DMRT, p ≤ 0.05).

Impact On Superoxide Radical (O), Hydrogen Peroxide, Lipid Peroxidation And Electrical Conductivity

The various oxidative stress markers viz., O₂^•−, H₂O₂, MDA and EC level were measured during Se and Si supplementation with or without As stress. The level of O₂^•− (51%), H₂O₂ (47%) and MDA (36%) were significantly enhanced with the exposure of As in comparison to the control. However, the level of these markers were reduced in As + Se, As + Si and As + Se + Si exposed rice plants with the maximum reduction being in As + Se + Si. The combination of Se + Si applied against As reduced the O₂^•− (28%), MDA (38%) as well as H₂O₂ (32%) in comparison to As alone treatment. In addition, the increase in EC upon As exposure (58%) was also reversed upon the combined supply of Se + Si, with the decline being 13% in comparison to As alone exposed plants (Fig. 1).

Antioxidant Enzymes Activities

The oxidative damage disrupts the plant’s biochemical and physiological responses. To protect against oxidative damages, plants contain a strong defence system of antioxidants. The first enzyme of antioxidant system is SOD whose activity was enhanced in the case of As alone exposure (60%) as compared to control (Fig. 2A). Upon application of Se + Si with As, the activity of SOD showed slight increase with the maximum increase being in As + Se + Si (36% in comparison to control). The CAT, APX and GPX activity significantly decreased upon As exposure (19%, 26% and 23%, respectively) as compare to control. Upon Se and Si alone supplementation, CAT, APX and GPX activity increased significantly (p ≥ 0.01) as compared to control and As alone exposure (Fig. 2B-D). These activities were also increased in Se + Si + As (71%, 67%, and 37%, respectively) as compared to the As alone exposure. The DHAR (Fig. 2E) activity showed no significant change in As alone treatment as compare with control. The DHAR activity showed the maximum increase in Se alone (67%) followed by Si alone (45%) treated rice plant as compared to control. However, supplementation of both Se and Si to As showed 22% decrease in the activity. The AOX activity was significantly increased by 44% against As exposure. However, the activity of AOX in As + Se + Si was reduced (19%) in comparison to As alone treatment.

Glutathione And Thiolic Metabolism Activity

GPx are key ROS-scavenging enzymes that catalyse H₂O₂ reduction to protect the cellular machinery from H₂O₂ damage. Plant GPx are the same as animal GPx, with the exception that their active domain contains cysteine rather than selenocysteine. Glutathione dependent enzymes i.e., GPx, GR and GST, activities were found to be significantly enhanced by 19%, 62% and 50% in As exposure as compared to the control (Fig. 3A-C). GPx activity was the maximum (25%) in Se alone exposed plant. The activity of these enzymes were significantly enhanced in As + Se and As + Si treatments also. In, As + Se + Si the activity of GPx and GR were increased by 45% and 29% as compared to As alone exposure.

CS, γ-ECS and SAT activities (Fig. 3D-F) were positively correlated with As accumulation. Results indicated that As exposure significantly (p < 0.05) elevated the activity of CS γ-ECS and SAT by 59% 19% and 42%, respectively as compared to control. Se and Si alone exposed plant enhanced the CS and SAT level. The maximum increase in CS and SAT activity was noticed in As + Se + Si exposure (80% and 38%, respectively) as compare to control. However, the activity of γ-ECS was decreased by 12% in As + Se + Si as compare to As alone exposure.

Effect Of Different Elements On Hacat Cell Lines

HaCaT cell lines supplemented with As showed decrease in cell viability. The percentage inhibition in viability of HaCaT cells lines treated with As was 31% as compared to control. The Se and Si alone treated HaCaT cell lines showed no significant change as compare to control. The Se + As and Si + As showed positive increase in growth of cells, which was 29% and 27% respectively, as compared to As alone treatment. The combination of Si + Se with As treated cell lines improved the cell viability (33%) as compared to As alone treatment (Fig. 4). Overall, results exhibited that the toxicity impact of As treatment on HaCaT cell lines was reduced by the single or combination of Se and Si treatments.

Measurement Of Cytomorphological Changes In The Hacat Cells

The morphological changes in the HaCaT cells were observed under phase-contrast microscopy (Fig. 5). The control (untreated) cells appeared to be uniformly spread and normal in surface with no distinctive or momentous changes in morphology even after 48 h of incubation. HaCaT cell lines growth showed the maximum decrease in As treatment (Fig. 5B). Some cells were found with their plasma membranes unblemished demonstrating that apoptosis had begun. The combination of As + Se and As + Si recovered the cell lines as compare to As alone. Arsenic induced shrinking and apoptosis in the cells occurred, which was recovered by Se + Si combination application.

Moreover, a strong correlation amongst various parameters of growth, accumulation, antioxidant enzymes, cell viability and other parameters were noticed in our study upon supplementation of Se and Si to As exposed rice plants. The correlation matrix among the different analysed parameters depicts a green colour indicating positive correlation (R² = 1) while red colour shows negative correlation. Other colours indicate non-significant correlation between different concentrations of various parameters (Fig. 6).

The reduction in the growth parameters of the rice seedlings under As stress is in agreement with earlier studies (Kumar et al. 2016, 2020; Copper et al., 2020; Dwivedi et al., 2020; Khan et al. 2021). Arsenic stress is well known to alter water uptake, diminish photosynthesis potential and affect metabolism of plants leading to decrease in growth and biomass accumulation (Yadav et al. 2021; Gupta et al. 2022). Lange et al. (2020) reported that As is non-significant for the plants and excluded from any biological activity of plants and therefore, it only negatively affects rice plant growth, development along with crop yield. In the present study, both elements induced growth in rice plants and reduced the toxic impact of As, which might be ascribed to antagonistic as well as competitive effects of Se and Si on As uptake. Selenium is also an essential element and a part of several seleno-proteins i.e. glutathione peroxidise and thioredoxin reductases (Feng et al. 2009; Malik et al. 2012; Bhadwal and Sharma 2022). Silica has been reported to enhance the growth and development of various plants (Tripathi et al. 2013; Emam et al. 2014; Dwivedi et al. 2020; Ismail et al. 2022). Si plays a crucial role and strengthens the rice stem and accumulated in conducting tissue. Hence, Se and Si supply improved growth of plants by serving their normal functions.

In addition, another possibility is that As accumulation in plants was also decreased due to competitive inhibition of As uptake (Ma et al. 2006; Afton et al. 2009; Gupta et al., 2020). In concordance with the previous reports, the present study showed a significant reduction in As uptake and translocation to the shoots in As + Se + Si treated rice coupled with increased growth parameters. Reduction in As accumulation in rice supplemented with Se and Si with the combination of As + Se, As + Si and As + Se + Si suggested competition for uptake and thus the reduction in As accumulation (Ma et al. 2008; Feng et al. 2009; Boorboori et al. 2021). Reduced As accumulation due to Si has also been reported by various authors (Tripathi et al. 2013; Dwivedi et al. 2020; Pan et al. 2021). Similarly, Kumar et al. (2014) also reported that the presence of Se decreases the As uptake in rice. Dwivedi et al. (2020) showed that the application of Si formulation effectively reduced As level even in highly As-contaminated soil. Interestingly, the combination of Se + Si application reduced more As level in rice as evidence by Alam et al. (2019). Thus, addition of Se + Si as a fertilizers will be a low cost way to reduce the toxicity in rice including grain grown in As contaminated sites and may be helpful in reducing human health risk associated with intake of As tainted rice.

Selenium induced plant growth and higher level of photosynthetic pigments in mungbean (Phaseolus aureus Roxb.) during As stress (Malik et al. 2012). The application of Se and Si with AsV boosted photosynthetic efficacy and pigment contents, according to Das and Biswas (2022).

Thiobarbituric acid–reactive substance is a by product of lipid peroxidation and can be used as bioindicator of toxicity. Increased TBARS content under As stress signified toxicity as reported by many authors (Kumar et al. 2013; Kumar et al. 2015; Dubey et al. 2018). The TBARS content in rice treated with Se and Si showed a mark reduction in the present study that suggested toxicity amelioration. The membrane damage reduction in terms of TBARS was also evident in decrease in EC in As + Se + Si treatment. The causal factor for TBARS might be H₂O₂, a reactive oxygen species (ROS), whose level was enhanced under As treatment. However, decreased level of H₂O₂ upon the application of Se and Si showed the reduced toxicity of As in the cell. This is similar to that of earlier studies (Tripathi et al. 2013; Kumar et al. 2014b; Dubey et al. 2018), where researchers observed the lower level of H₂O₂ in the presence of Se and Si in rice plants under As stresses. Thus, Se and Si supply augmented plants’ tolerance and growth through reduction in ROS, which might be achieved via changes in antioxidant system of plants.

To ascertain this, role of antioxidants was also evaluated. In plants antioxidant enzymes system acts as a secondary detoxification pathway and reduces oxidative stress stimulated through As. In As stressed plants, the activity of enzymatic antioxidants like SOD and GR, increased significantly. However, the activities of APX, GPX and CAT deceased in As-stressed plants in comparison to their respective control that might be due to As prompted inhibition of these enzymes. Antioxidant enzyme SOD is the converts O²⁻. to H₂O₂, which is further reduced to oxygen and water by APX in an ascorbate-glutathione cycle. GPX and CAT also play same function in the cytoplasm including other cellular compartments. The activity of enzymes was found to be augmented to a much higher level in Se + Si supplemented plants than in AsIII alone treatment. Glutathione transferases (GSTs) are known as ligands and affect the conjugation of the reduced GSH to contaminants (xenobiotic) for the purpose of detoxification. GSTs can operate in peroxidase and isomerase mode of action to reduce harmful effects of ROS. Our result showed increase of antioxidants in rice plants exposed to Se and Si amended As medium and well corroborated with earlier studies (Camera et al. 2019; Boorbori 2021). Current results showed marked increase in GPx and GST activities under As(III) stress. This might be due to GSH-dependent peroxide scavenging, which promoted reduction of oxidative damage, and membrane protecting role of Si through GST, preventing further damage of membrane.

Effect On Hacat Cell Lines

Heavy metal induced toxicity induces growth inhibition and cell death in plants and animals as well as humans have been reported by various authors. It has been reported that the As can promote the production of ROS, causing disturbances in the antioxidant system and damage to the cell wall rigidity (Lemarie et al. 2004; Gómez et al. 2005; Murugavel et al. 2007; Rahman et al. 2018; Ren et al. 2022). These ROS instigate cytotoxicity via mitogen activated protein kinases (MAPKs) along with tumor necrosis factor (TNF). These in turn harmonize various cellular functions like cell proliferation, differentiation, as well as apoptosis (Shen and Liu 2006; Rahman et al. 2018). Moreover, As also prompted cytotoxicity through tumor-suppressor protein 53 (p53) and nuclear factor-кB (NFкB), that ultimately modifies diverse process like cell growth regulation, and apoptosis (Wang et al., 2015). The decrease observed in HaCaT cell line growth under As stress was the result of direct toxicity associated with membrane breakdown, peroxide formation, ROS generation and series of biochemical alterations. Toxicity inside HaCaT cell line resulted into DNA damage, nuclear and cytoplasmic shrinkage, and chromatin fragmentation (Graham-Evans et al. 2004). The epithelial keratinocytes are the foremost cells to As exposure via inhalation, ingestion or through direct contact and are very useful to study the As mediated cytotoxicity. The As induced cytotoxicity using HaCaT cell lines provides a simulation of impact of As on biological system (Udensi, et al. 2011). However, co-application of Se and Si alleviated As induced cyto-toxicity on HaCaT cell lines. Selenium antagonistically alleviated As induced toxicity by altering cyto-genotoxicity and oxidative stress both in vitro and in vivo (Jamwal and Niyogi 2017; Rahman et al. 2018). Selenium acts as a natural antidote to As by increasing As excretion and as an antioxidant component of the enzyme glutathione peroxidase (Spallholz et al. 2004). Selenium and Si also stimulated growth responses in HaCaT cell lines in combination of As + Se and As + Si representing tolerance and antagonestic relationships of Se and Si against As reported by several authors (Spallholz et al. 2004; Kumar et al. 2014b, 2016; Dwivedi et al. 2020).

In conclusion, the As stress hampered the plant growth and this reduction was reversed with addition of Se and Si singly or in combination (Se + Si). The growth improvement was linked to reduce As accumulation in As + Se + Si along with improved enzymatic antioxidant system and decrease in MDA, DHA, ASC and H₂O₂ levels. In addition, cytotoxicity analysis of HaCaT cell lines with different elements revealed that the supplementation of Se + Si combination improved cell viability and their morphology. Thus, the combination of Se + Si fertilization may find application in reducing As accumulation and toxicity both in plant and human systems. Further, targeted field experiments and more animal/human model experiments would be required to take this approach to application.

Acknowledgements

The authors are thankful to Botany Department, Lucknow University, Lucknow for the facilities. Amit Kumar is thankful to SERB, DST, New Delhi for the award and financial assistance in form of SERB-NPDF.

Supplementary material: Supplementary file has been attached with this article.

Funding- No funding was received for conducting this study.

Competing Interest- The authors declare no competing interest.

Ethical approval- Not applicable.

Availability of data and materials- Not applicable

Consent to participate- Not applicable

Consent for publication- It is declared that the publisher has the author’s permission to publish the relevant contribution.

Author contributions- AK performed experimental work, prepared figures and writing original draft. SS, MIA and KG- writing– review & editing. PKS helped in biochemical analysis and AB and SK worked in cytotoxicity assay in HaCaT cells. All authors have read and approved the manuscript.

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Synergistic effects of selenium and silicon mitigate arsenic toxicity in plant (Oryza sativa L.) and human model (keratinocyte HaCaT cells lines) systems

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Abstract

Figures

Introduction

Material And Methods

Growth condition and experimental design

Plant Growth Measurement And Photosynthetic Pigments

Quantification Of Arsenic, Selenium, Silica And Quality Control

Specific Metal Accumulation And Translocation Factor

Estimation Of Superoxide Radical (O), Thiobarbituric Acid Reactive Substances Hydrogen Peroxide, And Electrical Conductivity

Estimation Of Oxidative Stress And Activity Of Antioxidant Enzymes

Cytotoxicity Assay In Hacat Cells

Cytomorphological Analysis By Phase-contrast Microscopy

Statistical analysis

Results

Impact on growth parameter and photosynthetic pigments

Arsenic, Selenium And Silica Accumulation

Impact On Superoxide Radical (O), Hydrogen Peroxide, Lipid Peroxidation And Electrical Conductivity

Antioxidant Enzymes Activities

Glutathione And Thiolic Metabolism Activity

Effect Of Different Elements On Hacat Cell Lines

Measurement Of Cytomorphological Changes In The Hacat Cells

Discussion

Effect On Hacat Cell Lines

Conclusions

Declarations

References

Supplementary Files

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