The product of NO2 ! was measured using the UV-Vis spectrophotometer at 540 nm. The content of H2O2 was determined after extraction by homogenizing plant tissues with 2 mL cold acetone . After centrifugation at 5000g at 4 ” C for 10 min, a 1.0 mL aliquot of the supernatant was mixed with 0.1 mL of 5% TiSO4 and 0.1 mL ammonia. After centrifugation, the titanium-peroxide complex pellet was resuspended in 3.0 mL of 2 M H2SO4, and absorbance was determined at 415 nm with a standard curve generated with known concentrations of H2O2.Plasma membrane integrity was evaluated by staining roots with Evans blue solution as in Yamamoto et al. with minor modifications. After the PPCP treatment, roots were stained with Evans blue solution for 15 min, and the stained roots were washed thoroughly with deionized water. The trapped Evans blue was released by extracting the roots in 5.0 mL of N,N-dimethylformamide. Absorbance of the supernatant was determined spectrophotometrically at 600 nm. The level of lipid peroxidation in roots and shoots of cucumber was measured in terms of malondialdehyde, which was determined according to the reaction with thiobarbituric acid as described in Yamamoto et al. . Historical staining for lipid peroxidation was conducted with Schiff’s reagent .For the measurement of reduced glutathione and oxidized glutathione , plant tissues were homogenized in 5 mL of cold 5% meta-phosphoric acid on ice. The homogenate was centrifuged at 12,000 g for 15 min at 4 ” C, and the supernatant was used for analysis of GSH and GSSG . A 0.5-mL aliquot of the supernatant was added to a reaction mixture containing 100 mM PBS , 0.2 mM NADPH and 1 mM 50 50 – dithiobis-2-nitrobenzonic acid . The reaction was started by the addition of 3 U of glutathione reductase, and absorbance at 412 nm was measured after 5 min. For GSSG, 2- vinylpyridine was added to the neutralized supernatant to mask GSH. Simultaneously,grow hydroponic the same volume of water was added for the total glutathione assay. The GSH concentration was obtained by subtracting the GSSG from the total GSH.Fresh plant tissue samples were frozen in liquid nitrogen, and homogenized in 50 mM PBS containing 1 mM EDTA and 1% PVP, with the addition of 1 mM ascorbate for ascorbate peroxidase.
The homogenate was centrifuged at 12,000 g for 20 min at 4 ” C, and the supernatant was used for the following enzyme assays . Protein content in enzyme extracts was determined by Coomassie brilliant blue G-250 with a standard curve using bovine serum albumin as the standard. Measurement of superoxide dismutase activity was carried out by inhibiting photochemical reduction of nitro blue tetrazolium . The assay mixture contained 50 mM PBS , 13 mM methionine, 75 mM NBT and 2 mM riboflavin. After addition of 100 mL of enzyme extract, the glass tubes were placed under light for 15 min, and then read at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT. To determine ascorbate peroxidase activity, a 200 mL aliquot of enzyme extract was added to the reaction mixture of 1 mM ascorbate and 0.3 mM H2O2 in 50 mM PBS. The absorbance changes were monitored at 290 nm for 3 min as ascorbate was oxidized, the enzyme activity was calculated using the extinction coefficient of 2.8 mM! 1 cm! 1 for ascorbate. Peroxidase activity was monitored by oxidation of 0.2% guaiacol using 0.3% H2O2 after addition of 50 mL enzyme extract. The enzyme activity was calculated using an extinction coefficient of 26.6 mM! 1 cm! 1 . Glutathione S-transferase activity was determined in 2 mL of a reaction mixture containing 50 mM PBS , 1 mM 1-chloro-2,4-dinitrobenzene , 5 mM GSH and 100 mL enzyme extract. The GST activity was measured spectrophotometrically at 340 nm based on GSH-CDNB adduct synthesis using extinction coefficient 9.6 mM! 1 cm! 1 for GSH-CDNB.To evaluate the sensitivity of cucumber plants to PPCPs, an initial dose-response experiment was carried out. Statistical analysis showed no significant difference in the biomass of plants grown in 0.5, 5 ng L! 1 PPCP-spiked and control solutions . However, treatment with PPCPs at 50 mg L! 1 progressively caused an increase of leaf necrosis . The level of chlorophyll a and chlorophyll b decreased with increasing PPCP treatment rates . Meanwhile, root activity decreased by 15.4% and 28.2% after exposure to 5 and 50 mg L! 1 PPCPs, respectively, as compared with the control .Previous studies showed that reactive oxygen species were produced when plants were exposed to xenobiotics .
However, little is known if exposure to PPCPs would induce intracellular ROS production in higher plants. It was found that even at 0.5 mg L! 1 , PPCPs induced H2O2 accumulation in both shoots and roots, and the maximum elevation occurred at the highest concentration , where H2O2 levels were about 3.0- and 3.2-fold higher in shoots and roots, respectively, than those from the control treatment . Additionally, O2 .- did not increase significantly after exposure to PPCPs at 0.5 mg L! 1 , which could be due to the fast conversion of O2 .- to H2O2.Analysis of Evans blue uptake and malondialdehyde content showed that PPCPs caused oxidative damage to the plasma membrane and lipid fraction in plant seedlings; however the damage was less pronounced in the leaves. A significant increase in Evans blue uptake was found at 50 mg L! 1 PPCPs in the root , and histochemical staining clearly indicated that cell death occurred in the root tip that is the most active and sensitive region of the root. Lipid peroxidation measured as MDA increased in all of the stressed plants, and its content was higher in roots than in shoots in all of the PPCP treatments . These results were further confirmed by histochemical analysis using Schiff’s reagent to detect lipid peroxidation in plants .After 7 d of cultivation, SOD showed the maximal activity in roots after exposure to PPCPs at 5 mg L! 1 and decreased thereafter . Ascorbate peroxidase showed a dose-dependent response, increasing about 2.0- and 1.1-fold in roots and shoots, respectively, after exposure to PPCPs at 50 mg L! 1 . Peroxidase is among the enzymes with a potential role in the detoxification of a variety of xenobiotics, and GSTs often detoxify exogenous compounds by conjugation with GSH. The total POD and GST activities both increased appreciably after exposure to PPCPs. The GSH content increased in leaves after exposure to PPCPs, while the root showed a maximal GSH content at 0.5 mg L! 1 PPCPs, followed by decreases thereafter . The decreases of GSH in roots may be due to GSH serving as an antioxidant for preventing oxidative damage, and also acting to detoxify PPCPs by conjugation. The GSSG content displayed little change when PPCP concentrations were low . However, when the PPCP concentration was increased to 50 mg L! 1 , there was a significant increase in GSSG content .Results from the present study illustrated the physiological, biochemical and molecular mechanisms involved in the detoxification of PPCPs in plants by considering especially homeostasis of ROS and anti-oxidant metabolism.
The results clearly showed that PPCP-induced morphological indicators changed at elevated PPCP concentrations , and the impact was more pronounced in roots than shoots. The enhanced sensitivity of roots to PPCP toxicity may be due to the greater accumulation of PPCPs in the root . Similar observations were previously reported in alfalfa, lettuce,and pepper after PPCP exposure . In addition, Christou et al. suggested that PPCPs in a mixture displayed a different uptake pattern compared with that when exposed individually. The present study also showed that roots consistently accumulated PPCPs to a higher level than shoots when exposed to mixed PPCPs . In shoots, growing lettuce hydroponically the relative accumulation of individual PPCPs differed somewhat from that in Wu et al. , where carbamazepine and diazepam were found at the highest levels, followed by meprobamate and trimethoprim. The discrepancy between the studies may be attributed to the different growth conditions, plant cultivars, and sampling intervals. Acetaminophen, however, was not detected in the plant tissues , likely owing to its rapid metabolism after uptake . Recent studies showed that contact with PPCPs was capable of inducing a complex set of physiological responses in higher plants . Generally, contents of leaf pigments, including chlorophyll and carotenoids, provide valuable information about the physiological status of a plant. Here, a clear leaf necrosis and reduction in contents of chlorophyll and carotenoids were observed at higher PPCP concentrations . Findings from this and previous studies together suggest that PPCPs may significantly affect plant growth. Prior to the induction of whole plant morphological effects, stress may also lead to physiological, biochemical and molecular changes within the plant. However, little information is available about the toxic effects in plants from a mechanistic perspective. A direct result of stress-induced cellular changes is the enhanced ROS accumulation, consequently imposing oxidative stress to bio-molecules . Although additional research is needed to establish oxidative stress as the primary mechanism of PPCP toxicity to higher plants, it is clear that oxidative stress is involved in the development of PPCP-induced toxic symptoms. In this study, changes in ROS levels were observed in comparison to the control after exposure to PPCPs , and the response occurred at much lower concentrations than that for morphological effects. Overproduction of ROS can cause cell damage and is the final consequence of oxidative stress. In the current study, the increase in ROS production coincided with the increase in membrane damage and lipid peroxidation in the cucumber plants, indicating the presence of oxidative stress. In a previous study, exposure to 10 mg L! 1 of diclofenac, sulfamethoxazole, trimethoprim or 17a-ethinylestradiol did not induce significant lipid peroxidation in alfalfa leaves . Exposure to mixed PPCPs was found to exacerbate cytotoxicity to a rainbow trout gonadal cell line as compared to exposure to individual compounds . These results indicated that studies using individual PPCPs might underestimate the actual environmental impacts of trace organic contaminants that usually occur as mixtures. During the period of time in which cucumber plants were exposed to the PPCP mixture, an overall induction of enzymatic and non-enzymatic antioxidant systems was observed. Superoxide dismutase constitutes the first line of defense against ROS, which can dismutate O2 .- into the more stable H2O2 . In this study, the root experienced more significant oxidative damage as a result of greater ROS accumulation than shoots, and elevated activities of SOD were observed in treatments below 5 mg L! 1 of PPCPs .
Above the 5 mg L! 1 treatment rate, SOD activity in roots was significantly inhibited. In a previous study,increased SOD activity was detected after exposure to paracetamol , while decreased SOD activity was found under triclosan and galaxolide stress in wheat seedlings. A possible explanation for the decreased SOD activity may be that the oxidative stress exceeded the capability of the enzymatic machinery. It must be noted that there exist three forms of SOD isoenzymes: copper/zinc containing SOD, manganese containing SOD and iron containing SOD, which are all localized in different cellular compartments. Further research should take into account the specific roles of different SOD isoenzymes from different cellular compartments after exposure to trace xenobiotics. Using ascorbate as a reductant, H2O2 was reduced to water by ascorbate peroxidase , the major component of the ascorbate-glutathione cycle . Thus the increase in APX activity in the PPCP treated cucumber plants may be related to the functioning of the ascorbate-glutathione cycle that detoxifies H2O2 thereby preventing further damage. This was consistent with previous observations in Brassica juncea with acetaminophen treatment . It has been shown that APX may be responsible for the fine modulation of ROS for signaling, whereas CAT might be responsible for the removal of excess ROS during stress. The significantly increased catalase activity with increasing PPCP concentrations reaffirmed that the cucumber plants experienced serious oxidative stress when in contact with the PPCP mixture. Beside anti-oxidation, another key role of POD and GSTs is their ability to inactivate toxic compounds . For example, Agostini et al. demonstrated the capacity of peroxidases to degrade the pesticide 2,4-dichlorophenol in a cell culture of Brassica napus. Xia et al. suggested that the induced activity of GST by chlorpyrifos indicated formation of glutathione S-conjugates to detoxify the insecticide in plants.