Metabolites were identified following the Metabolomics Standards Initiative conventions, using the highest confidence level , which is identified as at least two orthogonal measures versus authentic chemical standards . Three orthogonal measures were used to compare samples with authentic chemical reference standards: retention time , fragmentation spectra , and accurate mass . Peak height and retention time consistency for the LC/MS run were ascertained by analyzing quality control samples that were included at the beginning, during, and at the end of the run. Internal standards were used to assess sample-to sample consistency for peak area and retention times. Metabolite peak heights were scaled relative to the maximum peak height in any sample within an experiment to allow for relative comparison of peak heights between samples , but not for absolute metabolite level quantification.Chemical classes were assigned to metabolites with the ClassyFire compound classification system.To explore the variation between experimental conditions,vertical vegetable tower the metabolite profiles were PCA-ordinated, and the 95% confidence level was displayed as ellipses for each treatment. Hierarchical clustering analysis with a Bray–Curtis Dissimilarity Matrix was performed with the python 2.7 Seaborn package. Metabolite significance levels were analyzed with the Python SciPy ANOVA test coupled to a python Tukey’s honestly significant difference test with alpha = .05 corresponding to a 95% confidence level.
To test whether metabolites sorbed to clay were accessible for a plant-associated bacterium, the desorption rates of metabolites from different substrates were tested in a first experiment, and the growth rate of Pseudomonas fluorescens WCS415 on various substrates pre-incubated with metabolites was tested in a second experiment. The desorption rate of metabolites from substrates was determined for glass beads , sand , and clay . The substrates were incubated with 50 times concentrated defined medium or with 0× DM for 6 hr at 23°C. The substrates were subsequently washed three times with water, to remove soluble metabolites. The recovered metabolites of all three steps were analyzed by LC/MS, as described above. Substrates were added to a 12-well plate , and 2 ml of 0× DM was added to each well. A Pseudomonas fluorescens WCS415 preculture was grown in 5 ml 20× DM for 16 hr at 30°C, 200 rpm. The culture was pelleted at 4,000 g, 23°C for 5 min, and resuspended in 0× DM. The wells were inoculated with an initial optical density of 0.05 in triplicates. The plates were incubated at 30°C for 3 d , 1 ml of the supernatant was removed to determine OD at 600 nm. Positive growth controls were P. fluorescens grown in the same experimental setup in 50×, 20×, 10×, and 0× DM, but without substrate. A set of negative controls was prepared to account for different variables in the experiment: Substrates incubated with 0× DM with bacteria were set up as a growth control, accounting for metabolites already adsorbed to clay. Substrates incubated with 50× and 0× DM but without bacteria were used to control for changes in optical density of clay caused by DM.
The metabolite desorption experiment was performed by adding 2 cm3 of clay pre-incubated with 50× or 0× DM to a 12-well plate in triplicates, followed by the addition of 2 ml of 0× DM. The plate was incubated for 3 d at 30°C. Subsequently, 1.5 ml of the supernatant was removed by pipetting and placed in a new 12-well plate. Half of the wells were inoculated with P. fluorescens, the other half served as negative controls. OD at 600 nm was determined after 3 d of growth at 30°C.Global climate change has resulted in shifts in precipitation patterns, causing stress on freshwater resources, especially in arid and semi-arid regions . In many of these areas, demand for water has led to increasing use of municipally treated wastewater . Agriculture has been one of the primary targets for TWW reuse with water districts and governments promoting the adoption of recycled water for irrigation . However, the use of TWW for irrigation may come with potential risks, as TWW is known to contain a wide variety of human pharmaceuticals . The use of pharmaceutical compounds has increased with population growth and economic development, resulting in over 1500 compounds currently in circulation . Their widespread consumption has led to their occurrence in TWW as well as in TWW impacted surface water . For many of these pharmaceuticals, there is limited knowledge about their potential chronic effects in the environment . Further, many of these compounds can transform in the environment, resulting in the formation of transient or recalcitrant transformation products, many with unknown fates and effects in environmental compartments .
Diazepam belongs to the class of psychoactive compounds known as benzodiazepines, one of the most prescribed classes of pharmaceuticals . Diazepam is one of the most commonly detected pharmaceuticals in TWW, with concentration ranging from ng L−1 to low μg L−1 . This is likely due to its extensive use and low removal efficiency during secondary wastewater treatment . In humans, diazepam is primarily metabolized via phase I oxidative metabolism by demethylation to nordiazepam , or hydroxylation to temazepam , and then further oxidized to oxazepam . Oxazepam undergoes phase II metabolism via rapid glucuronidation and then excretion via urine . The three primary metabolites of diazepam are psychoactive compounds, and each is a prescribed pharmaceutical for treating psychological conditions and alcohol withdrawal symptoms . Both oxazepam and nordiazepam have been commonly detected in TWW, often at μg L−1 levels . However, there is little knowledge about the occurrence, formation, and fate of such metabolites outside the wastewater treatment systems . Several studies have focused on the uptake and accumulation of pharmaceuticals in agricultural plants as a result of TWW irrigation . These studies have demonstrated the capacity of higher plants to take up these compounds; however, until recently, relatively little consideration has been given to their metabolism in plants . Recent studies have shown that higher plants can metabolize xenobiotics similarly to humans with phase I modification reactions followed by phase II conjugation reactions using detoxification enzymes that function as a ‘green liver’ . In higher plants, phase I and phase II reactions are followed by a phase III sequestration, resulting in the formation of bound residues . Many of these studies have also highlighted a chemical-specific and species-specific nature of plant metabolism of pharmaceuticals. In this study, we examined the uptake and bio-transformation of diazepam in higher plants.Cucumber and radish seedlings were then used under hydroponic conditions to understand metabolism of diazepam and its effect on selected metabolic enzymes in whole plants.PSB-D A. thaliana cell line was purchased from the Arabidopsis Biological Resource Center at Ohio State University and cultured in a liquid culture suspension at 25 °C and 130 rpm in the dark.
Cell cultures were maintained in accordance with the ARBC maintenance protocol . The A. thaliana seed culture was produced by inoculating 7 mL of cell culture into 43 mL fresh growth media, followed by 96 h cultivation at 25 °C on a rotary shaker in the dark. After 96 h, 3 mL of the seed culture was inoculated into 27 mL fresh growth media to create an approximate initial cell density of 3.3 g . Flasks were spiked with 30 μL of a stock solution of diazepam and 10 μL of a 14Cdiazepam stock solution to yield an initial concentration of 1 μg mL−1 and a specific radioactivity of 7.4 × 103 dpm mL−1 with an initial methanol content of 0.13% . Simultaneously, control treatments were prepared by auto-claving cell suspension flasks before chemical spiking , flasks containing diazepam without cells , and flasks containing living cells without diazepam . Control treatments were used to determine adsorption,vertical farming equipments abiotic degradation, and potential toxicity to cells. Flasks were incubated for 120 h in triplicate and sacrificed at 0, 6, 12, 24, 48 and 96 h for sampling and analysis. At each sampling time point, samples were collected and centrifuged at 13,000g for 15 min in 50 mL polypropylene tubes. The supernatant was collected and stored at −20 °C until further analysis. Cells were immediately stored at −80 °C and then freeze-dried for 72 h. After drying, each sample was spiked with 50 μL of 10 mg L−1 diazepam-d5 as a surrogate for extraction-recovery calibration and extracted using a method from Wu et al. , with minor modifications. Briefly, cells were sonicated for 20 min with 20 mL methyl tert-butyl ether and then 20 mL of acetonitrile and centrifuged at 13,000g for 15 min. The supernatants were combined and concentrated to near dryness under nitrogen at 35 °C and then reconstituted in 1 mL of methanol. The cells were then extracted with 20 mL acidified deionized water and the supernatant was combined with the methanol extract for cleanup. Prior to clean-up, 100 μL of cell material extract and growth media were combined with 5 mL liquid scintillation cocktail I to measure the radioactivity in the extractable form on a Beckman LS500TD Liquid Scintillation Counter . Clean-up was carried out using solid phase extraction with 150 mg Waters Oasis© HLB cartridges that were preconditioned with 7 mL methanol and 14 mL deionized water. Samples were loaded onto cartridges and then eluted with 20 mL methanol under gravity. The eluate was dried under nitrogen and further recovered in 1.5 mL methanol:water . After re-suspension extracts were transferred to micro-centrifuge tubes and centrifuged at 12,000g in a tabletop d2012 Micro-Centrifuge . Samples were further filtered through a 0.22-μm polytetrafluoroethylene membrane into 2 mL glass vials and stored at −20 °Cbefore analysis. Extraction of growth media was done after adjusting the solution to pH 3 using HCl, and followed by SPE with Waters HLB cartridges, as described above. The extraction recoveries for the tissues and media were 88 ± 7% and 80 ± 14%, respectively. After extraction, the cell matter was air dried, and a 10-mg sub-sample was removed and combusted on a Biological Oxidizer OX-500 to determine the radioactivity in the non-extractable form. The evolved 14CO2 was captured in 15 mL Harvey Carbon-14 Cocktail II and analyzed on a LSC.Hydroponic cultivations were carried out using cucumber and radish seedlings.
Seeds were purchased from Lowes and germinated in a commercially labeled organic potting soil in a growth chamber . After the appearance of the first true leaf, uniform seedlings were selected, rinsed with distilled water, and individually placed in amber jars containing 900 mL hydroponic solution . After 3 d of adaption, plants were exposed to diazepam by spiking with 100 μL of the above stock solutions to reach a nominal concentration of 1 mg L−1 and an initial specific radioactivity of 2.5 × 103 dpm L−1 . The cultivation lasted for 7 d. A parallel treatment with an initial diazepam concentration at 1 μg L−1 and a specific radioactivity of 2.5 × 102 dpm was included to simulate more realistic exposure levels and to validate the high level treatments. The cultivation lasted for 28 d with the culture solution renewed every 3 d. Plant blanks and treatment blanks were placed alongside the treatment jars. At the end of 7 d or 28 d cultivation, the seedlings were removed from the jars. Before sample preparation, roots were rinsed thoroughly with distilled water. Harvested plants were separated into below ground biomass and above ground biomass . Flowering buds from cucumbers were also separated to observe any potential for accumulation in fruits. Tissues were freeze-dried and then stored at −80 °C until analysis. A 0.2-g aliquot of the dried plant tissue was ground to a fine powder using a mortar and pestle. Samples were extracted and prepared as described above. A six-point TCP standard calibration curve was used to determine activity.Active plant metabolism of diazepam was validated using a range of controls. No diazepam was detected in the non-treated media or the cell blanks, and there was no significant degradation of diazepam in the cell free media, suggesting no contamination or significant abiotic transformation. Moreover, no significant difference was seen in cell mass between the chemical-free control and the treatments, indicating that diazepam did not inhibit the growth of A. thaliana. Furthermore, no significant amount of diazepam was adsorbed to the cell matter in the non-viable cell control. In contrast, diazepam dissipated appreciably from the media containing viable cells, with the average concentration decreasing from 698 ± 41.5 to 563 ± 8.93 ng mL−1 after 120 h of incubation, a decrease of nearly 20% .