Antioxidant system of garden cress sprouts for using in bio-monitor of cadmium and lead contamination

Based on garden cress significantly used for phytoremediation, the antioxidant system included antioxidant-phenolic compounds and antioxidant-enzymes of 6-day-garden cress sprouts (GCS) were assessed as potential bio-indicators for cadmium (Cd) and lead (Pb) contamination. Total phenolic and flavonoid contents of GCS germinated under Cd and Pb treatments (25–150 mg kg−1) gradually increased with increasing concentration of metals and peaked by 2.0, 2.6, and 2.5, 2.3 folds at 150 mg kg−1, respectively. By using DPPH, ABTS, and PMC antioxidant assays, the total antioxidant activity of phenolic compounds of GCS increased 6.1, 13.0, and 5.8-fold for Cd and 5.9, 14.6, and 8.2-fold for Pb at 150 mg kg−1, respectively. The antioxidant enzymes of GCS (POD, CAT, GR, and GST) were significantly activated in response to Cd and Pb stress, and two new electrophoretic POD bands were detected. GCS was absorbed 19.0% and 21.3% of Cd and Pb at 150 mg metal kg−1, respectively. In conclusion, the approaches of the antioxidant defense system of GSC could potentially be used as bio-indicator for monitoring Cd and Pb contamination in a short time of germination process.

www.nature.com/scientificreports/ Determination of total flavonoid content. Methanol extract (250 µL), 5% NaNO 2 solution (75 µL), and distilled water (1.25 mL) were incubated at 25-30°C for 5 min. Aluminum chloride solution (10%) and NaOH solution (1.0 M) were then added to the mixture, which was maintained for 10 min at 25-30 °C. The absorbance of each mixture was recorded at 510 nm 26 . A catechin equivalent (CE) standard curve was used to calculate total flavonoid content, which was expressed as mg CE 100 g DW −1 .
ABTS assay. Methanol extract (10 µL) was added to 990 µL of ABTS (2,2-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) reagent and incubated for 1 min at 25-30°C 28 . The absorbance of the mixture was recorded at 734 nm. The ABTS radical-scavenging activity percentage was calculated using the formula given for the DPPH assay (above). The IC 50 value is the phenolic concentration required to scavenge 50% of either DPPH or ABTS free radicals.
PMC assay. A phosphor-molybdenum complex (PMC) antioxidant assay was conducted according to Prieto et al. 29 to determine antioxidant activity. The methanol extract (50 µL) was incubated with 950 µL of 28 mM sodium phosphate, 4 mM ammonium molybdate, and 600 mM sulfuric acid for 45 min at 90°C. The mixture was then cooled before absorbance was read at 695 nm. The EC 50 was considered the phenolic concentration equivalent to 0.5 OD at 695 nm.
Total antioxidant activity. Following the work of Abdel-Aty et al. 23 , total antioxidant activity was calculated as follows: total antioxidant activity = total phenolic content (mg GAE 100 g DW −1 )/mg IC 50 or mg EC 50 .
Preparation of crude enzyme extract. According to the method of Abdel-Aty et al. 23 , one gram of GCS was homogenized with 10 mL of extraction buffer (50 mM Tris-HCl, pH 7.0 containing 1 mM EDTA) using a glass mortar under cooled conditions. After centrifugation (12,000 g for 12 min at −4ºC), each supernatant (crude enzyme extract) was stored at −20ºC until further use.
Assays of antioxidant enzymes. Peroxidase (POD; EC 1.11.1.7) activity was estimated following the method described by Miranda et al. 30 . The assay was performed in 50 mM sodium acetate buffer (pH 5.5) in the presence of two substrates, 8 mM H 2 O 2 and 40-mM guaiacol, as well as crude enzyme extract. The increase in the absorbance at 1.0 per min at 470 nm was considered as one unit. Catalase (CAT; EC 1.11.1.6) activity was estimated using the method described by Bergmeyer 31 . The assay was performed in 75 mM phosphate buffer (pH 7.0) in the presence of 25 mM H 2 O 2 as a substrate as well as crude enzyme extract. The decrease in absorbance at 0.1 per min at 240 nm was considered as one unit. Glutathione-S-transferase (GST; EC 2.5.1.18) activity was estimated according to the method described by Habig et al. 32 The assay was conducted in 0.1 M potassium phosphate buffer (pH 6.5) and 1.6 mM GSH, with 1 mM CDNB added to the crude enzyme extract. The increase in absorbance was read at 340 nm. The enzyme concentration that converted 1 µM CDNB per min was considered as one unit. Glutathione reductase (GR; EC 1.6.4.2) activity was estimated using the method described by Zanetti 33 . The reaction was based on the oxidation of NADPH in the presence of GSSG as a substrate. The reaction mixture included 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, GSSG, NADPH, and crude enzyme extract. The enzyme concentration that oxidized 1 µmol of NADPH per min was considered as one unit.
Determination of peroxidase by gel electrophoresis. To identify peroxidase isozyme variations between the control and metal treatments of GCS, native polyacrylamide gel electrophoresis (native-PAGE) was performed according to the method of Stegemann et al. 34 with a slight modification. The substrate solution contained benzidine HCl (0.25 g), 4 mL of glacial acetic acid, and distilled water (added up to a total volume of 50 mL). After electrophoresis, the gel was placed into the substrate solution along with 10 drops of hydrogen peroxide. The gel was incubated at room temperature until bands appeared.
Determination of total heavy metals. Before metal determination, all samples were digested using nitric acid, an acceptable matrix for consistent recovery of metals that are compatible with the analytical method 35 . All heavy metal analyses were performed on an Agilent 5100 Inductively Coupled Plasma-Optical Emission Spectrometer with Synchronous Vertical Dual View. For each series of measurements, an intensity calibration curve was constructed that was composed of a blank and three or more standards from Merck (Germany). The accuracy and precision of the metal's measurements were confirmed using external reference standards from Merck; standard reference materials for trace elements in water and quality control samples from the National Institute of Standards and Technology were used to confirm the instrument reading. All experimental procedures were carried out in compliance with relevant guidelines.
Statistical analysis. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test; these tests were conducted in GraphPad Prism version 5. Data are presented as means ± SD (n = 4) and differences were considered significant at p < 0.01.

Results and discussion
Total phenolic and flavonoid contents. Table 1 screens the total phenolic and flavonoid contents of GCS grown in media contaminated with Cd and Pb at various concentrations (25-150 mg kg −1 ). The total phenolic and total flavonoid contents of untreated GCS as a control (1600 ± 51 mg GAE 100 g −1 DW and 231±6.6 mg CE 100 g −1 DW, respectively) significantly increased (p < 0.01) and reached their highest levels at the highest doses of Cd (3196±54 mg GAE 100 g −1 and 590±10.2 mg CE 100 g −1 DW, respectively) and Pb (3960 ± 76 mg GAE 100 g −1 and 522 ± 10.5 mg CE 100 g −1 DW, respectively) by 2.0, 2.6 and 2.5, 2.3-fold, respectively. Similarly, over-expression of phenolics and flavonoids was detected at 2.48-2.50-fold and 1.5-2.0-fold, respectively, above the levels in controls in C4 weed subjected to aluminum stress 36 . Accumulation of antioxidant-phenolic compounds in tissues facilitates the ability of plants to tolerate and detoxify Cd and Pb stress via their metal chelating activity and/or their antioxidant activity 37 . The hydroxyl and carboxylic groups of the phenolic compounds assist with the binding of metals. In addition, flavonoids, as an important class of antioxidant-phenolic compounds, aid the detoxification of ROS free radicals that are induced by heavy metal stress. Rice, as a cereal plant, responds to cadmium stress by accumulating antioxidant-phenolic compounds 38 . An increase in phenolic compounds is correlated with increases in Cd and Pb concentrations, which suggests that de novo synthesis of soluble phenolic compounds and/or hydrolysis of conjugated phenolic compounds occurs under heavy metal stress 39 . In addition, the increase in the soluble phenolic compounds that are used in the lignin biosynthesis of cell walls to create physical barriers against the harmful action of heavy metals has also been reported 40 . From such observations, we can conclude that the total phenolic and flavonoid contents of GCS are highly sensitive markers for indicating Pb and Cd contamination in soils, even when concentrations are low.

Antioxidant activities.
Most of the recently identified phenolic compounds of GCS have potent antioxidant activity 23 . Therefore, the antioxidant activity of GCS germinated in media contaminated with Cd and Pb was evaluated by various antioxidant assays. In Table 2, IC 50 values using DPPH and ABTS methods and EC 50 values using the PMC method of untreated GCS (0.0098, 0.0065, and 0.007 mg GAE mL −1 , respectively) gradually and significantly decreased (p < 0.01) and reached to their lowest values (0.0041, 0.0011 and 0.0021 mg GAE mL −1 , respectively) at highest concentrations of Cd and Pb, respectively. Low IC 50 and EC 50 values reflect a high antioxidant activity. Additionally, the total antioxidant activity of GCS gradually and significantly increased relative to the control levels (p < 0.01) to reach maximum levels for Cd by 6.1-, 13.0-, and 5.8-fold and for Pb by 5.9-, 14.6-, and 8.2-fold at the highest concentrations of Cd and Pb (Table 3). This increase in antioxidant activity may be attributed to increases in the concentration of antioxidant-phenolic compounds that were associated with increasing Cd and Pb concentrations in growth media. ABTS and DPPH free radicals were tested in all antioxidation reactions of organic residues that were combined with ROS radicals 41,42 . Therefore, the antioxidant activities of GCS could be used as potential bioindicators for Cd and Pb toxicity. In previous studies, the antioxidant activity of rice was found to increase during Cd stress, which facilitated metal chelating and enhanced Cd tolerance 38 . Total antioxidant activity increased from 1.2-to 1.7-fold in Malva parviflora roots and leaves under different Cd concentration treatments 43 . Additionally, ABTS and DPPH free radical scavenging activity as well as PMC reduction activity gradually increased in C4 weed grown under aluminum treatments 36 . In contrast, chickpeas grown under different heavy metals treatments had an antioxidant activity that remained lower than that of the control, according to DPPH or ABTS assays, but that slightly increased as the concentration of accumulated metal increased 44 .
Antioxidant enzymes. Cd and Pb can cause the overproduction of ROS free radicals and induce oxidative damage to plant tissues. Maintaining the balance between ROS free radicals and activation of the antioxidative www.nature.com/scientificreports/ system under heavy metal stress is a critical protective mechanism in plants that diminishes oxidative damage in polluted tissues 15 . Therefore, in the present work, the potential role of the antioxidant enzymes (POD, CAT, GR, and GST) in response to oxidative stress in GCS under Cd and Pb treatments was investigated; the results are presented in Fig. 1. POD activity gradually increased (p < 0.01) with increasing of Cd and Pb concentrations till reached highest activity (355 and 451 U g −1 , respectively) compared to the control (153 U g 1 ) (Fig. 1A).
The results observed a strong correlation between the phenolic content and POD activity of GCS germinated under Cd and Pb treatments. POD is involved in removing H 2 O 2 as a harmful ROS-free radical by oxidation of phenolic compounds [45][46][47][48] . Similarly, increasing Hg accumulation in garden cress shoots was correlated with an increase in POD activity and changes in total carotenoid content 19 . Furthermore, POD participates in the polymerization of phenolic compounds for lignin synthesis to build a barrier that protects against toxic metal ions 49 .
In the antioxidant defense system, CAT also stopped the hyper-accumulation of H 2 O 2 by converting it to water and oxygen. Here, compared with control CAT activity (100 U g −1 ), an increase in Cd and Pb treatment concentrations caused a significant increase (p < 0.01) in CAT activity, which peaked (250 and 190 U g −1 , respectively) at 75 mg metal kg −1 (Fig. 1B). Similarly, CAT and POD activities were significantly enhanced in vetiver grass, Populus nigra, Cajanus cajan [50][51][52] , and Malva parviflora 43 grown in Cd-and/or Pb-contaminated soils. This considerable increase in CAT activity could be induced by excessive production of H 2 O 2 under 75 mg Cd and Pb kg −1 . However, the CAT activity was reduced under higher concentrations of Cd and Pb (100 and 150 mg kg −1 ), which may be due to severe oxidative damage occurring under much higher Cd and Pb levels. The severity of the Cd/Pb stress may suppress enzyme synthesis or alter the enzyme's assembly 53 . Interestingly, this decline in CAT activity was compensated by the increase in POD activity to remove H 2 O 2 . Many previous investigations on some plant species have revealed that variations in antioxidant enzyme activities were related to the severity of Cd/Pb stress 53,54 .  www.nature.com/scientificreports/ The important GSH-utilizing enzymes, GST and GR, were also evaluated in the current study. These enzymes largely participate in the efficient metabolism of ROS free radicals and their products 55 . Hence, they tightly control heavy metal-induced oxidative stress in plants. The activities of GST and GR enzymes significantly www.nature.com/scientificreports/ increased relative to control levels (239 and 900 U g −1 , respectively) in all Cd and Pb treatments, and the activities reached their peak at 75 metal mg kg −1 treatments (Cd: 700 and 566 U g −1 , respectively; Pb: 2,330 and 2,318 U g −1 , respectively) ( Fig. 1C and D). An increase in GST activity was previously reported in pumpkin, rice, and Cicer arietinum in response to either Cd or Pb stress 17,56,57 . Our results revealed a strong correlation between the phenolic compound levels and GST activity of GCS under Cd and Pb treatments, suggesting that GST not only removes toxic ROS radicals but also transports the phenolic compound chelating-metal complexes to the vacuoles 58 . In a previous study, GR activity increased in Brassica napus leaves under Cd stress 59 . In contrast, GR activity decreased in Ceratophyllum demersum and mung bean seedlings in response to Cd stress 60,61 . Such variations in GR activity and responses may have been due to differences in plant genotypes 58 . In the results described above, there was a direct relationship between Cd and Pb concentrations and POD, CAT, GST, and GR activities, which indicates that these four enzymes of the antioxidant defense system could be used as bio-indicators for Cd and Pb stress. In addition, the GCS can cope with the oxidative damage induced by Pb and Cd.
Electrophoretic pattern of peroxidase activity. Fig. 2 shows the electrophoretic patterns of peroxidase in GCS germinated under Cd and Pb treatments (25-150 mg kg −1 ) compared with untreated controls. Two peroxidase isozymes were detected in untreated control and the intensity of these bands increased gradually with increasing Cd and Pb concentrations; moreover, two new peroxidase isozymes appeared. This observation explains why POD activity gradually increased in all Cd and Pb treatments to cope with ROS that caused stressrelated damage to plant tissues.  (Fig. 3). Thus, GCS seem to have the ability to take up Cd and Pb from contaminated media as phytoremediator in a short germination period (6 days). A previous study reported that garden cress plants could absorb (through their shoots and roots) 48% of Pb from soil contaminated with a 300-mg kg −1 concentration after a 30-day culture 20 . Another study found that garden cress   www.nature.com/scientificreports/ reduced 100-mg kg −1 Hg by 33% in contaminated growth media after six repeated phytoextraction processes 19 . Additionally, chickpea seedlings produced maximum uptake of Pb (~3.8%) from contaminated soil at a Pb concentration of 250 mg kg −144 .

Bioaccumulation of Cd and
Visual changes of GCS and germination index. During six days of germination, the GCS did not exhibit any noticeable visual changes when subjected to different concentrations of Cd and Pb, even under substantially higher concentrations of both. In addition, the germination index (GI) of the GCS was conducted throughout 6 days of germination under different Cd and Pb concentrations (25-150 mg/kg), as seen in Fig. 4, and the results showed that the GI was unaffected by these heavy metals even at their higher doses. One could conclude that the potent antioxidant defense system of GCS could easily counter the damaging effects of Cd and Pb toxicity. Some plants exposed to toxic levels of Cd and Pb decrease germination and interfere with seedling physiological processes 62 such as cowpea, soybean, lettuce, and sugar beet 8 . While parsley seedlings that grew under considerably higher Cd concentrations didn't show any signs of visual changes 63 .
Bio-indicators collective data. All of the tested parameters that were measured in GCS germinated under the lowest Cd and Pb concentrations (25 mg kg −1 ) observed fold increase values of 1.5-2.2-fold (Table 4). At the highest Cd and Pb concentrations (150 mg kg −1 ), the fold-increase values of these parameters were slightly higher (1.7-2.9-fold), but the expected substantial increase did not occur, except for total antioxidant activity (5.8-14.6-fold) ( Table 4). These observations suggest that all the tested parameters of GCS responded strongly to lower concentrations of Cd and Pb and the antioxidant activity of phenolic compounds of GCS can be considered a potent bioindicator for monitoring Cd and Pb at both low and high concentrations. It is important to conduct these experiments outdoors in varied soils under different environmental conditions in order to study more realistic changes in the GCS-antioxidant system and the accumulation and distribution of these heavy metals.

Conclusion
In the present work, GCS was germinated with different concentrations of Cd and Pb (25-150 mg kg −1 ) under in vitro conditions. The phenolic and flavonoid levels and antioxidant capacity were enhanced several folds and correlated with the Cd and Pb concentrations. The activities of antioxidant enzymes (POD, CAT, GR, and GST) were strongly increased under Cd and Pb stress. In addition, new peroxidase isozymes appeared in the electrophoretic profile. Further, considerable amounts of Cd and Pb were absorbed by the tissues of GCS.  . Data are presented as means ± SD (n = 4) and differences were considered significant at p < 0.01. Table 4. Collective data on the fold-increase among all the tested parameters at the lowest and highest concentrations of Cd and Pb in comparison to their controls. Data are presented as means ± SD (n = 4) and values in the same column with different superscripts were considered significant at p < 0.01.  www.nature.com/scientificreports/