Enantioselective uptake and translocation of atenolol in higher plants.

The presence of pharmaceuticals in surface water and wastewater has been an increasing area of research since they can represent a possible route for human exposure when these waters are used to irrigate crops. The concentration of these drugs in crops depends on their uptake and translocation within plants. A less recognized question is that over 50 % of pharmaceuticals are chiral compounds, but there is little knowledge about their enantioselectivity in plants. In this study, we evaluated the uptake, bioconcentration, and translocation of enantiomers of atenolol, a commonly used beta-blocker, in Arabidopsis thaliana cells and Lactuca sativa plants under hydroponic conditions. Atenolol was taken up by Arabidopsis thaliana cells during 120 h of exposure to solutions with 1 mg/L of R/S-(±)-atenolol. A moderate preference for R-(+)-atenolol over S-(-)-atenolol was observed, with the enantiomeric fraction (EF) reaching 0.532 ± 0.002 for the R enantiomer. Atenolol was also taken up and translocated by Lactuca sativa after hydroponic cultivation in nutrient solutions containing 1 or 10 µg/L R/S-(±)-atenolol. Moderate enantioselectivity was detected in the treatment with 10 µg/L, and the EF after 168 h was 0.42 ± 0.01, suggesting that S-(-)-atenolol was preferentially accumulated. Selectivity was also observed in the translocation factor (TF), calculated as the ratio of the concentration in the leaves over that in the roots. As many emerging contaminants are chiral, our findings highlight the importance to consider their fate and risks in terrestrial ecosystems at the enantiomer scale.

Effect of low-dose, repeated exposure of contaminants of emerging concern on plant development and hormone homeostasis.

Treated wastewater is increasingly used to meet agriculture's water needs; however, treated wastewater contains numerous contaminants of emerging concern (CECs). With exposure and uptake of CECs, phytotoxicity and health of crop plants is of concern, but is poorly understood. This study evaluated the effect of low-dose, chronic exposure to a mixture of 10 CECs, including 4 antibiotics, 3 anti-inflammatory drugs, 1 antiepileptic, 1 beta-blocker, and 1 antimicrobial, on lettuce (Lactuca sativa) and cucumber (Cucumis sativa L.) plants. The CEC mixture was added in nutrient media at 1 to 20X of their typical levels in treated wastewater effluents. Biological endpoints including germination, growth, phytohormone homeostasis, and CEC bioaccumulation were determined. Exposure to the CEC mixture did not affect the germination rate of lettuce seeds, but stimulated root elongation and increased the root-to-shoot biomass ratio during a 7 d cultivation. A dose-dependent decrease in biomass was observed in cucumber seedling after a 30 d exposure, with the highest rate CEC treatment resulting in decreases of 51.2 ±â€¯20.9, 26.3 ±â€¯34.1, and 33.2 ±â€¯41.7% in the below-ground, above-ground, and total biomass, respectively. Levels of abscisic acid were significantly elevated (p < 0.05) in the leaves, but decreased (p < 0.05) in the roots. The dose-response of auxin was characterized by a hormesis effect. A significant 6-fold increase in the stem auxin level was observed at the 1X CEC rate, followed by a decrease to 2-fold the control at the 20X rate. Leaf auxin concentrations also significantly increased at the 1X CEC rate to 16-fold, followed by a decrease at the highest CEC rate. The results of this study suggeste that chronic exposure to low levels of CEC mixtures may compromise the fitness of plants, and the impairments are underlined by alterations in hormone balances.

Formation of biologically active benzodiazepine metabolites in Arabidopsis thaliana cell cultures and vegetable plants under hydroponic conditions.

The use of recycled water for agricultural irrigation comes with the concern of exposure to crops by contaminants of emerging concerns (CECs). The concentration of CECs in plant tissues will depend on uptake, translocation and metabolism in plants. However, relatively little is known about plant metabolism of CECs, particularly under chronic exposure conditions. In this study, metabolism of the pharmaceutical diazepam was investigated in Arabidopsis thaliana cells and cucumber (Cucumis sativus) and radish (Raphanus sativus) seedlings grown in hydroponic solution following acute (7 d)/high concentration (1 mg L-1), and chronic (28 d)/low concentration (1 µg L-1) exposures. Liquid chromatography paired with mass spectrometry, 14C tracing, and enzyme extractions, were used to characterize the metabolic phases. The three major metabolites of diazepam - nordiazepam, temazepam and oxazepam - were detected as Phase I metabolites, with the longevity corresponding to that of human metabolism. Nordiazepam was the most prevalent metabolite at the end of the 5 d incubation in A. thaliana cells and 7 d, 28 d seedling cultivations. At the end of 7 d cultivation, non-extractable residues (Phase III) in radish and cucumber seedlings accounted for 14% and 33% of the added 14C-diazepam, respectively. By the end of 28 d incubation, the non-extractable radioactivity fraction further increased to 47% and 61%, indicating Phase III metabolism as an important destination for diazepam. Significant changes to glycosyltransferase activity were detected in both cucumber and radish seedlings exposed to diazepam. Findings of this study highlight the need to consider the formation of bioactive transformation intermediates and different phases of metabolism to achieve a comprehensive understanding of risks of CECs in agroecosystems.

Evaluation of the estrogen receptor alpha as a possible target of bifenthrin effects in the estrogenic and dopaminergic signaling pathways in zebrafish embryos.

Bifenthrin (BF) is a pyrethroid insecticide widely used in urban and agricultural applications. Previous studies in embryos of zebrafish have shown that BF can affect estradiol biosynthesis and the dopaminergic system. To examine the role of the estrogen receptor (ER) in the endocrine effects of BF, embryos were exposed for 96 h to a mixture of 0.15 and 1.5 µg/L BF and an ER agonist (17α-ethynylestradiol - EE2) at 0.09 µg/L. Transcripts related to estrogenic (vitellogenin VTG) and dopaminergic (tyrosine hydroxylase (TH), dopamine receptor 1 (DR1), monoamine oxidase (MAO), and catechol-O-methyltransferase b (COMTb)) signaling pathways were investigated by qRT-PCR. Dopamine (DA) and its metabolites (homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC)) were also measured. There was a significant increase in VTG, DR1, MAO and COMTb mRNA levels and HVA-DA ratios within all zebrafish embryos exposed to EE2, including EE2 alone, 0.15 µg/L BF + EE2 and 1.5 µg/L BF + EE2. A significant decrease in homogenate concentrations of DA was observed within all zebrafish embryos exposed to EE2, which included EE2 alone, 0.15 µg/L BF + EE2 and 1.5 µg/L BF + EE2. Co-exposure of BF with EE2 failed to diminish estrogenic or dopaminergic signaling in embryos. Additionally, embryos with diminished ERα expression by morpholino injection were exposed to 0.15 µg/L BF, 1.5 µg/L BF and 0.09 µg/L EE2, with subsequent gene expression measurements. ERα knockdown did not prevent the effects of BF, indicating ERα may have a limited role in the estrogenic and dopaminergic effects caused by BF in zebrafish embryos.

Acetaminophen detoxification in cucumber plants via induction of glutathione S-transferases.

Many pharmaceutical and personal care products (PPCPs) enter agroecosystems during reuse of treated wastewater and biosolids, presenting potential impacts on plant development. Here, acetaminophen, one of the most-used pharmaceuticals, was used to explore roles of glutathione (GSH) conjugation in its biotransformation in crop plants. Acetaminophen was taken up by plants, and conjugated quickly with GSH. After exposure to 5 mg L-1 acetaminophen for 144 h, GSH-acetaminophen conjugates were 15.2 ±â€¯1.3 nmol g-1 and 1.2 ±â€¯0.1 nmol g-1 in cucumber roots and leaves, respectively. Glutathione-acetaminophen was also observed in common bean, alfalfa, tomato, and wheat. Inhibition of cytochrome P450 decreased GSH conjugation. Moreover, the GSH conjugate was found to further convert to cysteine and N-acetylcysteine conjugates. Glutathione S-transferase activity was significantly elevated after exposure to acetaminophen, while levels of GSH decreased by 55.4% in roots after 48 h, followed by a gradual recovery thereafter. Enzymes involved in GSH synthesis, regeneration and transport were consistently induced to maintain the GSH homeostasis. Therefore, GST-mediated conjugation likely played a crucial role in minimizing phytotoxicity of acetaminophen and other PPCPs in plants.

Comparisons of analytical chemistry and biological activities of extracts from North Pacific gyre plastics with UV-treated and untreated plastics using in vitro and in vivo models.

Plastic debris is an emerging worldwide threat to marine biota. Marine species may face unique challenges in low-flow estuarine systems with a high abundance of "macro-sized" (>4.75 mm) plastic due to the leaching of constituents and adsorbed contaminants. To simulate this leaching process, plastic samples recovered from the North Pacific Gyre along with corresponding UV-irradiated virgin plastic and non-irradiated virgin plastic counterparts were incubated in saltwater for 30 days at ambient temperatures ranging from 17 to 25 °C. Following solid-phase extraction, water samples were fractionated with sequential methanol elution from 10 to 100% and evaluated using in vitro assays assessing estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) activities. In vivo responses (vitellogenin [vtg] and cytochrome p450 1A [cyp1a] mRNA) were measured following 5-day exposures in Japanese medaka (Oryzias latipes) larvae (3 days post hatch). Estrogenic plasticizers, co-planar PCBs and PAHs were quantified in the extracts using targeted GC-MS/MS and UPLC-MS/MS. In vitro estrogenicity showed highest activity in the 70% methanol fraction for all plastic leachate exposures. Whole extract in vitro estradiol equivalent (EEQ) values were 4.34 ±â€¯2.65, 8.79 ±â€¯2.09 and 13.78 ±â€¯3.64 ng/L, for virgin plastic, UV-irradiated virgin plastic and North Pacific Gyre-recovered plastic, respectively (mean ±â€¯SD). Significant vtg induction was observed in medaka larvae exposed to leachate extracts from North Pacific Gyre-recovered plastic and UV-irradiated virgin plastic (9.9-fold, p = 0.039 and 10.1-fold, p = 0.042, respectively). Chemically-determined EEQ values were also localized in the 70% methanol fraction. Whole leachate extract chemical EEQ values were 0.33 ±â€¯0.07, 1.64 ±â€¯0.62 and 11.4 ±â€¯2.13 ng/L, for virgin plastic, UV-irradiated virgin plastic and North Pacific Gyre-recovered plastic, respectively. In-vitro AhR activity was highest in the 70% methanol elution with greater activity in North Pacific Gyre-recovered plastic than in virgin plastic and UV-irradiated virgin plastic (toxic equivalency [TEQ] = 1.06 ±â€¯0.54, 0.38 ±â€¯0.07 and 0.71 ±â€¯0.47 ng/L, respectively). CYP1A mRNA was significantly induced in larval medaka exposed to North Pacific Gyre-recovered plastic leachates (17.8-fold, p = 0.02) while exposure to virgin plastic and UV-irradiated virgin plastic leachates caused no significant change. Chemically-determined TEQ analysis for AhR indicated highest activity in the 90% methanol fraction for all leachates, with whole extract in vitro TEQs being 1.47 ±â€¯0.87, 0.03 ±â€¯0.05 and 0.42 ±â€¯0.38 ng/L for North Pacific Gyre-recovered plastic, virgin plastic and UV-irradiated virgin plastic, respectively. These results indicate that weathering and UV radiation release estrogenic plasticizers and demonstrate the ability for plastics to transport adsorbed persistent organic pollutants at eco-toxicologically relevant concentrations.

Hydrogen peroxide mediates triclosan-induced inhibition of root growth in wheat seedlings.

Triclosan, an extensively used antimicrobial agent, enters agroecosystems when sewage sludge and reclaimed water are applied to agricultural fields, and may trigger a series of plant physiological and biochemical responses. However, few studies have investigated the mechanism by which plant development is affected by triclosan. Here, microscopic, pharmacological and biochemical analyses, and histochemical dye staining were used to explore the effects of triclosan on root growth in wheat plants. Exposure to triclosan inhibited root elongation, and significantly triggered hydrogen peroxide (H2O2) production and lipid peroxidation in wheat roots. The inhibition of root growth by triclosan was reversed by dimethylthiourea, a H2O2 scavenger, indicating that alterations of endogenous H2O2 concentrations in root cells were likely linked to triclosan-induced root growth inhibition. The addition of butylated hydroxyanisole, a lipophilic antioxidant, during triclosan treatment completely prevented the increase of lipid peroxidation, but did not alleviate triclosan-induced reduction of root growth. In triclosan-treated wheat roots, the level of indole-3-acetic acid decreased by 68.3%, while the contents of two indole-3-acetic acid oxidative metabolites, indole-3-aldehyde and indole-3-carboxylic acid, increased by 71.3% and 314.4%, respectively. Moreover, the oxidation of auxin induced by triclosan in wheat roots was prevented by dimethylthiourea. These results together suggested that the triclosan-enhanced production of H2O2 induced auxin oxidation, thus leading to the suppression of root growth. Findings of this study improve our mechanistic understanding on how antimicrobial agents such as triclosan affect plant root growth.

Stable Isotope Labeling-Assisted Metabolite Probing for Emerging Contaminants in Plants.

Biotransformation is a notable modulator of the fate, bioaccumulation, and toxicity of contaminants in the environment. However, it is often formidable to identify unknown biotransformation products in the absence of reference standards, and this analytical challenge is particularly true for contaminants of emerging concern (CECs) that are mostly polar molecules without characteristic structures (e.g., Cl and Br) and in complex matrices such as plants. In this study, using the fibrate drug gemfibrozil as a model CEC and Arabidopsis thaliana as a model plant, we developed and demonstrated a novel analytical framework coupling deuterium stable isotope labeling with high-resolution mass spectrometry (SILAMS) in identifying plant biotransformation products. When exposed in A. thaliana cells, gemfibrozil was quickly taken up into the cells and extensively metabolized. The use of nonlabeled and deuterated gemfibrozil at a 3:1 ratio created unique diagnostic patterns in mass spectra, enabling the identification of 11 novel phase II amino acid/peptide conjugates. Similarity in mass fragmentation patterns and chromatographic behaviors was then employed to establish the probable structures. Two major metabolites were further confirmed as glutamate and glutamine conjugates using authentic standards. Most of the identified conjugates were also detected in the whole A. thaliana plant. Therefore, SILAMS offers unique advantages by excluding false matrix positives and helping discern unknown metabolites, including polar conjugates with endogenous biomolecules, with a high degree of confidence. This novel framework may be readily applied to other CECs for high-throughput metabolite screening in plants to improve our understanding of their food safety and human health risks and potential deleterious effects on other species living on plants.

Metabolism of sulfamethoxazole in Arabidopsis thaliana cells and cucumber seedlings.

Reclaimed water is a historically underutilized resource. However, with increased population growth and global climate change, reclaimed water is evolving into an economical and sustainable water resource to meet the needs of citizens, industries, and agriculture. The use of recycled water for agricultural irrigation comes with the potential risk of environmental and food contamination by pharmaceuticals and personal care products (PPCPs). The levels of PPCPs in plants will depend on translocation and metabolism in plant tissues. However, relatively little is known about the metabolism of PPCPs in plants. In this study, the metabolism of the antibiotic sulfamethoxazole was investigated in Arabidopsis thaliana cells as well as cucumber seedlings grown under hydroponic conditions. Using high-resolution mass spectrometry and 14C tracing allowed for sulfamethoxazole metabolism to be comprehensively characterized through all metabolic phases. Six phase I and II metabolites were identified in A. thaliana cell cultures and cucumber seedlings. Sulfamethoxazole metabolism followed oxidation and then rapid conjugation with glutathione and leucine. Direct conjugation with the parent compound was also observed via acetylation and glucosylation. At the end of 96 and 168 h incubation, N4-acetylsulfamethoxazole was the major metabolite and >50% of the radiolabeled sulfamethoxazole became non-extractable in both A. thaliana cells and cucumber seedlings suggesting extensive phase III metabolism and detoxification. The study findings provided information for a better understanding of the uptake and metabolism of sulfamethoxazole in higher plants, highlighting the need to consider metabolic intermediates and terminal fate when assessing the risk of PPCPs in the soil-plant continuum.

Degradation of Deepwater Horizon oil buried in a Florida beach influenced by tidal pumping.

After Deepwater Horizon oil reached the Florida coast, oil was buried in Pensacola Beach (PB) sands to ~70cm depth, resulting in Total Petroleum Hydrocarbon (TPH) concentrations up to ~2kg per meter of beach. This study followed the decomposition of the buried oil and the factors influencing its degradation. The abundance of bacteria in oiled sand increased by 2 orders of magnitude within one week after oil burial, while diversity decreased by ~50%. Half-lives of aliphatic and aromatic hydrocarbons reached 25 and 22days, respectively. Aerobic microbial oil decomposition, promoted by tidal pumping, and human cleaning activities effectively removed oil from the beach. After one year, concentrations of GC-amenable hydrocarbons at PB were similar to those in the uncontaminated reference beach at St. George Island/FL, and microbial populations that disappeared after the oil contamination had reestablished. Yet, oxihydrocarbons can be found at PB to the present day.

Pharmaceutical and personal care products-induced stress symptoms and detoxification mechanisms in cucumber plants.

Contamination of agricultural soils by pharmaceutical and personal care products (PPCPs) resulting from the application of treated wastewater, biosolids and animal wastes constitutes a potential environmental risk in many countries. To date a handful of studies have considered the phytotoxicity of individual PPCPs in crop plants, however, little is known about the effect of PPCPs as mixtures at environmentally relevant levels. This study investigated the uptake and transport, physiological responses and detoxification of a mixture of 17 PPCPs in cucumber seedlings. All PPCPs were detected at higher concentrations in roots compared to leaves, with root activity inhibited in a dose-dependent manner. At 5-50 µg/L, the mature leaves exhibited burnt edges as well as a reduction in photosynthesis pigments. Reactive oxygen species (ROS) production and lipid peroxidation increased with increasing PPCP concentrations; and their contents were greater in roots than in leaves for all PPCP treatments. Enzymes involved in various functions, including oxidative stress (superoxide dismutase and ascorbate peroxidase) and xenobiotic metabolism (peroxidase and glutathione S-transferase), were elevated to different levels depending on the PPCP concentration. Glutathione content gradually increased in leaves, while a maxima occurred at 0.5 µg L-1 PPCPs in roots, followed by a decrease thereafter. This study illustrated the complexity of phytotoxicity after exposure to PPCP mixtures, and provided insights into the molecular mechanisms likely responsible for the detoxification of PPCPs in higher plants.

Contaminants of emerging concern affect Trichoplusia ni growth and development on artificial diets and a key host plant.

Many countries are utilizing reclaimed wastewater for agriculture because drought, rising temperatures, and expanding human populations are increasing water demands. Unfortunately, wastewater often contains biologically active, pseudopersistent pharmaceuticals, even after treatment. Runoff from farms and output from wastewater treatment plants also contribute high concentrations of pharmaceuticals to the environment. This study assessed the effects of common pharmaceuticals on an agricultural pest, Trichoplusia ni (Lepidoptera: Noctuidae). Larvae were reared on artificial diets spiked with contaminants of emerging concern (CECs) at environmentally relevant concentrations. Trichoplusia ni showed increased developmental time and mortality when reared on artificial diets containing antibiotics, hormones, or a mixture of contaminants. Mortality was also increased when T. ni were reared on tomatoes grown hydroponically with the same concentrations of antibiotics. The antibiotic-treated plants translocated ciprofloxacin through their tissues to roots, shoots, and leaves. Microbial communities of T. ni changed substantially between developmental stages and when exposed to CECs in their diets. Our results suggest that use of reclaimed wastewater for irrigation of crops can affect the developmental biology and microbial communities of an insect of agricultural importance.