A common pathway in most organisms for detoxifying xenobiotics is through glutathione conjugation catalyzed by glutathione Stransferases . It has long been recognized that GSTs play an important role in normal cellular metabolism in plants . In addition, GST mRNA responds quickly to exposure of xenobiotics , protecting the cell against oxidative stress or chemical toxicity . Previous research on xenobiotics such as herbicides shows that differences in GSH availability and in the portfolio of GST isoenzymes are associated with xenobiotic resistance . Conjugation with various biomolecules was observed in plant metabolism of different PPCPs, including diclofenac , ibuprofen , triclosan , benzotriazole and acetaminophen . However, GST-mediated conjugation may play a predominant role, but has been relatively understudied so far . In particular, the entire process of GST mediated detoxification, from GSH conjugation in the cytoplasm to vacuolar accumulation and processing of conjugates has yet to be elucidated in vivo. Acetaminophen and structural analogs are phenolic compounds that are among ubiquitous environmental contaminants . Acetaminophen is one of the most used pain and fever relief medicines, and many billions of doses of acetaminophen are consumed each year . Acetaminophen was detected in the aquatic environment at 0.01–0.3 mg L−1 in the South Wales region of the UK .Cucumber is a crop plant, widely consumed, with high economic and ecological relevance,hydroponic grow system and recommended for use in phytotoxicity studies by U.S. Environmental Protection Agency . In this study, we used acetaminophen to elucidate the mechanisms of GST-mediated detoxification in cucumber plants.
Findings of this study may provide evidence for PPCP detoxification via GSH conjugation, and will likely prompt further exploratory investigation on the role and value of this pathway for the numerous emerging contaminants.Cucumber seeds were obtained from Fisher and transferred to a 72-hole plate filled with a mixture of vermiculite and perlite . Plants were cultivated under controlled conditions for 1 week and irrigated with half strength modified Hoagland nutrient solution, which is adequate for the seedlings’ energy demands for cucumber growth and the low ionic strength nutrient solution permits a more precise computation of acetaminophen speciation than do more complete nutrient medium. Details on the solution composition are given in Table S2. Seedlings in the third-leaf stage were removed from the pots, rinsed with tap water, and placed in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution. Each treatment contained three replicates, and each replicate was prepared from a single plant by separating into roots and shoots.After 7 days of adaption, plants were exposed to acetaminophen by spiking 25 μL stock solution to yield an initial concentration of 5.0 mg L−1 .Simultaneously, treatment blanks and plant blanks were included in duplicate to determine abiotic losses of acetaminophen under the experimental conditions. All experimental materials were auto-claved, 121 °C for 30 min, before use. A parallel group trial enrolled with acetaminophen at 60 μg L−1 was included to simulate more realistic levels and to validate the high level treatment. The cultivation lasted for 20 days, with the nutrient solution renewed every other day . Additionally, in order to determine if plants can detoxify acetaminophen via GSH conjugation in realistic field environment, cucumber seedlings were cultivated for 20 days in soil amended with 5% bio-solids containing acetaminophen at an initial concentration of 10 mg kg−1 .
A root exudate control was included to evaluate the effect of plant exudates on microbial degradation and GSH conjugation of acetaminophen in the hydroponic solution. Cucumber seedlings were cultivated in 500 mL glass jars containing aerated, half strength modified Hoagland nutrient solution, and the plants were removed after 7 days. The solution was brought back to 500 mL with half strength modified Hoagland nutrient solution, and then 25 μL of acetaminophen stock solution was added to yield an initial concentration of 5.0 mg L−1 . The nutrient solution samples were exposed to the same conditions, and were analyzed for acetaminophen and its conjugates. To investigate whether GSH-acetaminophen can be transported from the root to the shoot, two-week-old cucumber plants were exposed to 1 mg L−1 GSH-acetaminophen for 2 days. Leaves were collected and extracts from the aerial parts were analyzed as described below. To test whether cytochrome P450 was involved in GSH-conjugates formation, two P450 inhibitors, 1-aminobenzotriazole and piperonyl butoxide, were used to pretreat the plant on the basis of previous studies demonstrated specific inhibition effects . Cucumber seedlings were pretreated with 1- aminobenzotriazole or piperonyl butoxide for 12 h in 500 mL glass jars with aerated, half strength modified Hoagland nutrient solution, followed by spiking 25 μL of acetaminophen stock solution to yield an initial concentration of 5.0 mg L−1 . After 96 h incubation, tissue samples were collected from both the treated and untreated plants.The specificity of these P450 inhibitors was evaluated by monitoring cytochrome P450 activity, GSH content and GST activity in cucumber tissues after the treatment. For better clarity, all the experiments performed was schematically represented Table S3. Simultaneously, commercially available common bean , tomato , alfalfa and wheat seeds were germinated and transplanted under the same conditions as above. After 7 days of adaption, plants were exposed to acetaminophen at 5.0 mg L−1 . After 96 h, plant roots were collected and analyzed for acetaminophen and its glutathione, cysteine and N-acetylcysteine conjugates.
Sampling, sample preparation and analysis Cucumber seedlings treated with or without 5 mg L−1 acetaminophen were sacrificed at 0, 12, 24, 48, 72, 96, 120 and 144 h. Before sample preparation, roots were rinsed thoroughly with tap water. Harvested tissues were separated into roots and shoots and then frozen in liquid nitrogen. Enzyme activities of GST, glutathione reductase , γ-glutamylcysteine synthetase , and cytochrome P450, as well as contents of reduced glutathione in both roots and shoots, and lipid peroxidation were immediately analyzed after sampling, as described below. The remaining plant samples were freeze-dried and stored at −80 °C until chemical analysis. At each sampling time, nutrient solution samples and the aqueous root exudate samples were collected and mixed with equal volumes of methanol. The mixture was added with d4-acetaminophen as a surrogate, filtered through a PTFE syringe filter and stored at −20 °C before analysis. To quantify acetaminophen and its glutathione,indoor garden cysteine and Nacetylcysteine conjugates, the freeze-dried plant tissue samples were ground to a fine powder with a mortar and pestle, and a 0.5-g aliquot was used for solvent extraction. Each sample was spiked with d4- acetaminophen as the recovery surrogate, and extracted, sequentially, with 20 mL methyl tert-butyl ether , acetonitrile, and 0.5 mM HCl solution in an ultrasonic water bath for 20 min for each extraction. The supernatant from MTBE and acetonitrile extraction was combined after centrifugation and reconstituted in 1.0 mL methanol after drying under nitrogen . The extract was pooled with the supernatant from the HCl extraction. The pooled extract was loaded onto an Oasis™ HLB cartridge that was preconditioned with 6 mL methanol and 12 mL deionized water, and eluted with 15 mL methanol under gravity. The eluate was evaporated to dryness under N2, and the residue was recovered in 1.5 mL methanol:water mixture and filtered before analysis. The instrumental analysis was performed on a Waters ACQUITY ultra-performance liquid chromatography coupled to a Waters Micromass Triple Quadrupole mass spectrometer equipped with an electrospray ionization interface . Acetaminophen, and its glutathione, cysteine and N-acetylcysteine conjugates were separated using ACQUITY UPLC BEH C18 column with water and methanol as mobile phases. Electrospray ionization was operated in the positive mode. Detailed information on the instrumental analysis is given in Table S4.Fresh cucumber roots were frozen in liquid nitrogen, and homogenized on ice with 5 mL of 50 mM potassium phosphate buffer containing 1 mM EDTA and 1% polyvinylpyrrolidone. After centrifugation at 15,000 g at 4 °C for 20 min, the supernatant was used for the assay of activities of glutathione S-transferase, glutathione reductase, and cytochrome P450 . Measurement of GST activity was carried out spectrophotometrically after the glutathione and 1-chloro-2,4-dinitrobenzene adduct synthesis reaction . Briefly, GST activity was measured in 2 mL of a reaction mixture containing 50 mM PBS , 1 mM CDNB, 5 mM GSH and 100 μL enzyme extract. The increase in absorbance as a result of GSH-CDNB synthesis was determined at 340 nm for 5 min on a Cary 50 UV– Visible spectrophotometer . The GST activity was calculated using the extinction coefficient of GSH-CDNB .
To determine the activity of GR , a 200 μL aliquot of enzyme extract was added to the reaction mixture of 0.15 mM NADPH and 0.5 mM oxidized glutathione in 50 mM PBS . The absorbance decrease was monitored at 340 nm for 3 min after GSSG-dependent consumption of NADPH . For the assay of γ-ECS , plant tissues were homogenized and suspended in 5 mL of 0.1 M HCl. After centrifugation at 20,000 g for10 min at 4 °C, the supernatant was collected. The γ-ECS activity was measured according to Shan and Liang . An incubation solution was prepared with 800 μL 50 mM TrisHCl containing 0.25 mM glutamate, 10 mM ATP, 1 mM dithioerythritol, 2 mM cysteine, and 400 μL of the γ-ECS sample. The mixed solution was incubated for 1 h at 25 °C, and then 800 μL phosphorus agent was added and mixed. After incubation at 45 °C for 25 min, the absorbance at 660 nm was measured on the UV–Visible spectrophotometer. One unit of γ-ECS activity was defined as 1 mmol cysteine-dependently generated PO4 3− per minute. The levels of proteins in the enzyme extracts were determined using Coomassie Brilliant Blue G-250 . Brieflfly, 100 μL of enzyme extract was added to 5 mL of 0.01% Coomassie Brilliant Blue G- 250. After 5 min, the absorbance at 595 nm was measured on the UV– Visible spectrophotometer. The protein content of enzyme extracts was calculated by comparison with a standard curve using bovine serum albumin as the standard. The protein concentrations in the extracts were estimated using the BSA standard curve. The content of GSH was determined spectrophotometrically according to Jiang et al. . Plant tissues were homogenized in 5 mL of cold 5% meta-phosphoric acid on ice and centrifuged at 12,000 g at 4 °C for 15 min, and the supernatant was analyzed for GSH. To 0.5 mL of supernatant, 0.5 mL PBS and 0.5 mL of 5′5′-dithiobis-2- nitrobenzonic acid were added. After thorough mixing and incubation for 5 min, the absorbance at 412 nm was measured . Membrane lipid peroxidation, a typical indicator of stress phytotoxicity , was estimated by measuring the concentration of malondialdehyde, according to the reaction with thiobarbituric acid as described in Sun et al. .All treatments were performed in triplicates. A procedural blank and a sample duplicate were included in every batch of 10 samples to monitor background contamination and reproducibility, and the calculated relative standard deviations were b10%. The d4-Acetaminophen was used to account for any loss during sample preparation, matrixinduced ionization effects, and variations in instrumental response. Authentic standards were used to confirm the target analytes. A six-point calibration line was used for quantification with the r 2 values of at least 0.99 for all analytes . No acetaminophen or any of the conjugates was detected in the solvent or treatment blanks. Limits of detection and quantification for individual acetaminophen and the conjugates were calculated as 3 and 10 times the signal-to noise level from the low-level spiked samples . The recoveries in all samples were within acceptable limits ranging from 75 to 110%, demonstrating good method accuracy and precision. Extraction efficiency was N91% for acetaminophen and its conjugates. The plant position was regularly rearranged every 2 days to avoid side-effects.The level of acetaminophen in the nutrient solution without plant, after the removal of plant , or with cucumber plant, all decreased over time . Significant losses occurred in the unplanted control or the root exudate control . Considering that the cultivation system was not a sterile environment, both microbial and abiotic degradation may have contributed to the apparent acetaminophen dissipation in the solutions. This observation was consistent with Bartha et al. , who also reported the occurrence of plant-independent, biotic processes that decreased the available acetaminophen in plant solution.