N4- acetylsulfamethoxazole was detected in A. thaliana cells and confirmed using its authentic standard. The glucose and glutathione conjugates were detected by comparing the exact mass and fragmentation patterns to proposed spectra libraries for each compound . However, observed difference in the fragmentation patterns indicated that conjugation location differed from in those observed for human metabolism. A similar pattern was observed in previous studies concerning the plant metabolism of pharmaceuticals . The proposed amino acid conjugate in A. thaliana cells is, to the best of our knowledge, the first evidence for their occurrence in higher plants. Conjugation with amino acids has been considered a detoxification pathway for other pharmaceuticals . The structure proposed here for leucylsulfamethoxazole was, in part, based on a m/z of 132.0765, 223.1135 and 255.1677 showing distinct fragments of C6H13NO2 C12H18N2 and C10H12N3O3S . The position of the amino acid on the benzene ring was selected based on optimum stable formation . In the tentative metabolism pathways in A. thaliana cells, sulfamethoxazole underwent Phase I oxidation,raspberries for containers forming 4-nitroso-sulfamethoxazole followed by phase II conjugation with leucine or glutathione. Based on the signal strength, a relatively high level of the N5-leucyl-sulfamethoxazole conjugate was detected at the 3 h sampling point but remained at trace levels for most of the incubation duration, with the exception of the 48h sampling point.
The glutathione conjugate appeared quickly , spiked at 48h, and decreased to a non-detectable level by the end of the cultivation . Conjugation with glucuronic acid was also observed to form quickly , and direct glycosylation of sulfamethoxazole has also been observed in mammals . Another pathway appeared to be acetylation of the sulfamethoxazole amine followed by rapid oxidation to form the S296 and S313 metabolites , with N4-acetylsulfamethoxazole being the predominant metabolite at the end of incubation. It has been shown that this acetylation pathway predominates in human metabolism for detoxification . Future research should be conducted to determine if enzymes similar to those seen in human metabolism of sulfamethoxazole actively participate in its metabolism in plants, such as CYP2C9 and N-acetyltransferases 1 and 2. Similar metabolites were found in cucumber seedlings grown in the nutrient solution containing sulfamethoxazole. Metabolites from Phase I and Phase II metabolism were similarly detected and extensive Phase III sequestration was further observed . When cucumber plants were exposed to 1.0 µg L-1 sulfamethoxazole in nutrient solution, sulfamethoxazole was taken up into the plant, with accumulation primarily in the roots, old leaves, and stems, and absent in the new leaves . When 14C activity was used for calculation, a translocation factor , i.e., the ratio of the concentration in leaves/stems over that in the roots, was estimated to be 0.32. While this value was higher than that reported by Dodgen et al. it suggested that sulfamethoxazole is not readily translocated in the plant after entering the root. This was consistent with models used to predict the behavior, e.g., diffusion through cell membranes, of similar pharmaceutical compounds .
These models suggested that polar compounds , such as sulfamethoxazole, would have translocation factors from the solution to the xylem ranging from 0.25 to 0.5. Bio-concentration factors were calculated for the roots and the shoots as the ratio of specific radioactivity in the tissue over that in the growth media. The mean BCFR and BCFS were determined to be 1.59 and 0.53, respectively. The calculated BCFR and BCFs were similar to those found for other vegetable species in Dodgen et al. . These studies together suggested that while plants are capable of taking up sulfamethoxazole, it mainly remains in the root with limited potential for translocation to the other organs of the plant . After the 7 d cultivation, sulfamethoxazole parent and metabolites identified in the A. thaliana cell incubation were similarly scanned in cucumber samples from the hydroponic cultivation experiment, including plants tissues and blank control without plants. The proposed metabolites were not detected in the control treatment or the cucumber hydroponic solution, indicating that the transformation occurred within the cucumber seedlings following uptake.However, due to the lack of authentic standards for most of the metabolites or low signal , we did not attempt to quantify individual metabolites in the cucumber plants. In a previous study, Chen et al. investigated the uptake, metabolism, and elimination of sulfamethoxazole in Brassica rapa chinensis and Ipomoea aquatica. In that study, no metabolites of sulfamethoxazole were detected in plant tissue. This could be attributed to a number of factors, such as, differences in extraction protocols, instrument analysis or extensive phase III metabolism that decreased the level of metabolites below the limit of detection. Our findings were in line with previous research conducted with the relatedsulfa-drug sulfamethazine, in which N-acetyl-sulfamethoxazole and hydroxysulfamethoxazole were detected in Zea mays L. plants .
The total amount of extractable and non-extractable residues in the cucumber plants ranged from 94% to 80% indicating that some mineralization occurred. This rate of mineralization in the plant cultivation system was higher than that of sulfamethoxazole in soils. Because plant respiration may contribute to the loss of 14C after mineralization, this finding further highlights the detoxification prowess of higher plants .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,blueberries in pots 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 biotransformation of diazepam in higher plants. Arabidopsis thaliana cells were used for an initial kinetic evaluation and metabolic profiling . 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 14C-diazepam 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, autoclaving cell suspension flasks prepared control treatments before chemical spiking , flasks containing diazepam without cells , and flasks containing living cells without diazepam . Control treatments were used to determine adsorption, 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 13,000 g 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 freezedried 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 13,000 g 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 clean-up. 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 microcentrifuge tubes and centrifuged at 12,000 g 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 °C before 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.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.