Demethylation is a common phase I metabolism typically catalyzed by cytochrome P450 enzymes that are ubiquitous in humans, terrestrial organisms and aquatic organisms. Sometimes demethylation can also occur via the catalysis of esterase or non-enzymatic hydrolysis for -COOCH3 and result in the formation of carboxyl groups.Methylation of CECs, on the other hand, is a phase II metabolism typically catalyzed by methyltransferases.Various substrates are susceptible to the activity of methyltransferases, such as nucleic acids, lipids and many xenobiotics.Unlike other phase II metabolism, methylation usually leads to increased hydrophobicity, but it is considered a detoxification pathway in most cases.64 Methylation and demethylation are among the most common transformations for CECs in the environment. For example, triclosan was methylated during the WWTP treatment, and the derived methyl triclosan was frequently detected in TWW along with triclosan, sometimes at even higher concentrations.Acetaminophen was reported to methylate during microbial degradation in soil. TBBPA monomethyl ether and dimethyl ether were frequently detected in the environment along with TBBPA, sometimes at higher concentrations. TBBPA MME and TBBPA DME were also formed through abiotic methylation in the natural presence of methyl iodide in aquatic environments.Biotic methylation of TBBPA was also observed to occur through biologically mediated transformations in sediments,earthworms ,vertical tower planter and plants. Methylation of BPA was promoted by Mycobacterium strains like PYR-1 and PCP1.Methylation of diclofenac was observed in aquatic invertebrates.
Demethylation of common CECs has been previously reported as well, such as the O-demethylation of naproxen in humans, terrestrial plants, microbes and soils, and the N-demethylation of diazepam in humans, terrestrial plants and aquatic organisms.Several studies have also shown the demethylation of methylated CECs back to the parent compound. For example, methyl triclosan was back converted to triclosan in A. thaliana and fish.Demethylation of TBBPA DME and TBBPA MME back to TBBPA was observed in pumpkin plants. The back conversion of diclofenac methyl ether in aquatic invertebrates was also reported.As an important type of TPs, methylated CECs are usually more hydrophobic than their corresponding parent compounds, and therefore may pose increased ecological risks. For example, consistently higher concentrations and BCFs of methyl triclosan, as compared with triclosan, were observed in fish, snails and algae collected from TWW-impacted streams.BPA mono- and dimethyl ether were more toxic to the development of zebrafish embryos than BPA itself.Diclofenac methyl ether showed greater bioaccumulation and further higher acute toxicity to H. azteca and G. pulex than diclofenac.However, exceptions exist to this general rule. For instance, the methyl ethers of TBBPA were less toxic to zebrafish development and earthworms in terms of acute exposure.Lower bio-accumulation factors of methyl triclosan than triclosan was reported in algae .These studies suggested that changes in environmental behaviors of CECs induced by methylation were molecule-specific. The investigation of back-and-forth conversion between methylation and demethylation of CECs is important to obtain a more complete understanding of the environmental cycling of CECs. This previously neglected conversion circle implies prolonged persistence of such contaminants in the environment. This transformation circle needs to be further investigated for more comprehensive and accurate risk assessment for CECs that are susceptible to such reactions.
In addition, research is needed to quantitatively evaluate differences in non-target toxicity between CECs and their methylated or demethylated derivatives. Although methylation and demethylation of several CECs in agroecosystems and aquatic invertebrates were studied previously,our overall knowledge of the transformation potential, fate and ecological risks of these methylated or demethylated TPs is limited. Many CECs contain active functional groups such as hydroxyl, methoxyl, carboxyl, ester and amide groups in their chemical structures, making them susceptible to methylation or demethylation under biotic and abiotic conditions. However, the specific molecular properties that promote methylation or demethylation need to be better understood. For example, methyltransferases in plants are involved in many important metabolic activities;therefore, the similarity in chemical structures to the endogenous biomolecular substrates may influence the potential for methylation of xenobiotics in plants. The bond strength of the chemical bond connecting the methyl group and the major fragment may also affect the potential for demethylation of CECs. Furthermore, methylated or demethylated TPs may be demethylated or methylated back to the parent CECs, respectively.This previously ignored metabolic circle may effectively prolong the persistence of CECs in the environment and lead to unrecognized environmental risks. Changes in environmental behaviors induced by methylation or demethylation are poorly understood with few experimental observations. Methylation of CECs can lead to increases in bio-accumulation, and acute and developmental toxicity in organisms,while exceptions also exist.Demethylation of CECs also does not necessarily lead to lower bio-accumulation and toxicity.Methylation and demethylation may also affect the transport, translocation and persistence of CECs by inducing changes in their hydrophobicity, solubility and pKa. The fate of demethylated and methylated TPs of CECs in agroecosystems and aquatic environments warrants a more systematic evaluation.
Given the large and ever-increasing number of CECs in the environment, it is unrealistic to experimentally investigate all CECs, let alone their methylated and/or demethylated TPs.The incorporation of modeling tools, such as QSARs and models based on the use of molecular descriptors, can help predict environmental behaviors and provide an alternative way to assessing the risks of CECs and their TPs. Such modeling approaches need to be validated and refined using experimental data. In conclusion, changes in the environmental behaviors of CECs induced by simple transformation reactions such as methylation and demethylation need to be systematically explored through rigorously designed experiments. The experimental data should be further incorporated into existing models to help validate the utility of models and also allow the prediction for a wide range of CECs for which experimental data may never be available. Contaminants of emerging concern refer to contaminants that are recently discovered in the environment and may pose potential adverse effects, such as developmental toxicity and endocrine disruption, to non-target organisms and human health at environment-relevant concentrations. Because of their widespread use, CECs are ubiquitously present at trace levels in treated wastewater and bio-solids. Many CECs contain reactive functional groups such as hydroxyl, carboxyl, and amide, making them susceptible to biotic and abiotic transformations during treatment at wastewater treatment plants. Therefore, in addition to the parent form of CECs, transformation products are also often present in treated wastewater and bio-solids, sometimes at even higher concentrations . Treated wastewater and bio-solids have been increasingly applied to agricultural lands in recent years in beneficial reuse practices, which serves as a conduit for plants to be contaminated with CECs and their TPs, posing potential human health and ecological risks . Methylation and demethylation are among the most common transformation reactions for many CECs. Biotic demethylation is a phase I metabolism process facilitated mainly by cytochrome P450 enzymes that are ubiquitous in organisms. For example, as a common pharmaceutical itself, nordiazepam is also a demethylated metabolite of diazepam excreted after oraladministration in humans . Likewise, demethylation can convert naproxen to O-desmethyl naproxen , and methylparaben to 4-hydroxybenzoic acid through microbially mediated phase I metabolism. Abiotic demethylation of herbicides and some CECs was also observed after advanced oxidation processes during wastewater treatment. Therefore,lettuce vertical farming demethylated counterparts are among the most commonly observed TPs of CECs. Biotic methylation is a phase II metabolism mediated by methyltransferases. Methylated acetaminophen, i.e., pacetanisidide , is a major metabolite of acetaminophen in soil. Methyl triclosan is the primary TP of the antimicrobial triclosan after WWTP treatment. Tetrabromobisphenol A , a brominated flame retardant, was found to be O-methylated by microbes, as well as in pumpkin plants and earthworms. The addition or loss of a methyl group during transformations alters a compound’s physicochemical properties 31, which may subsequently affect its fate and risk in the environment. As uptake of CECs into plants is known to depend closely on a chemical’s physicochemical properties, such as lipophilicity, it may be hypothesized that methylation or demethylation changes a chemical’s behavior and fate in the soil plant continuum.
Despite their frequent occurrence, the environmental significance of simple transformation reactions such as methylation and demethylation is often overlooked. In this study, we compared plant accumulation and translocation of four pairs of compounds differing only in a methyl group in their structures using two plant models, Arabidopsis thaliana cells and wheat seedlings. Four CECs and their respective methylated or demethylated counterparts were chosen as the test compounds because of their widespread use and occurrence in the environment . Of these compounds, DM-diazepam is not only a TP of diazepam, but also a pharmaceutical itself, and DM-methylparaben is not just a TP of methylparaben, but also an industrial raw material. Results from this study contribute to a better understanding of the implications of simple transformation reactions such as methylation and demethylation on the environmental behavior and potential risks of CECs.All analytical standards were purchased with reported purities ≥ 98%. Acetaminophen, diazepam, DM-diazepam, d5-diazepam , naproxen, DM-methylparaben and methylparaben were purchased from Sigma-Aldrich . M-Acetaminophen was purchased from Santa Cruz . DM-naproxen and d4-methylparaben were purchased from Toronto Research Chemicals . d4-Acetaminophen and d3-naproxen were purchased from C/D/N Isotopes . HPLC-grade methanol, acetonitrile, methyl tert-butyl ether and acetone were purchased from Fisher Scientific . Ultrapure water was generated by an in-house Milli-Q water purification system . Radioisotope labeled compounds were not used in this study, and therefore the uptake efficiency or mass balance of the target compounds in plants could not be derived. The A. thaliana cell suspension was obtained from the Arabidopsis Biological Resource Center at Ohio State University and was maintained in the laboratory at 24 °C and 130 rpm in NT-1 media with constant light . An aliquot of the A. thaliana cell culture was added to fresh, autoclaved NT-1 media , and incubated for 3 d, after which each cell suspension was spiked with individual compounds to arrive at an initial concentration of 1 mg/L. Control treatments included positive and negative control groups containing CECs spiked in nonviable cells , CECs in blank culture solution, or viable cell culture solution without CECs. Each treatment was prepared in triplicate, and was sampled at 1, 3, 6, 11, 24, 48, and 96 h. Entire samples were transferred to polypropylene centrifuge tubes and were immediately centrifugated at 3500 rpm for 30 min. The cell matter was stored at -80 ˚C until further analysis, and the supernatant was transferred into a 40 mL glass bottle and stored at -20 ˚C until further analysis.Wheat seedlings used in this study were germinated from seeds to avoid potential background contamination. Sterilized seeds were germinated on a moist filter paper on a tray in the dark at room temperature. The tray was then transferred into a growth chamber after 2 d for seedlings to grow. When the seedlings grew to about 5 cm in height, they were transplanted into a 50-mL polypropylene centrifuge tube wrapped in aluminum foil and then cultivated in the growth chamber. Initially filled with water, the solution in the tubes was replaced to 1/4 strength and then 1/2 strength Hoagland® nutrient solution at 2-day intervals to allow wheat seedlings gradually acclimating to the nutrient media. Once acclimated, the media were replaced with 30 mL fresh 1/2 strength Hoagland® nutrient solution spiked with a single compound of interest from individual stocks to reach a nominal chemical concentration of 1 mg/L. Water was added to each tube every other day to make up the lost water throughout the incubation experiment. Triplicate containers were sacrificed at 0, 3, 6, 12, 24, 48, 96, 168 and 240 h after the treatment. Plants were rinsed with deionized water, dried with paper towels, and separated into roots and shoots. The nutrient solutions remained in the centrifuge tubes and separated plant tissues were stored at -80 ℃ until further analysis. Transpiration stream concentration factors of target compounds were not measured in wheat seedlings in this study, due to challenges in collecting adequate amount of xylem sap for analysis.Deuterated compounds were used as surrogates during extraction for QA/QC. Extraction of nutrient solutions from A. thaliana cells and wheat seedlings was carried out using a similar method to a previous study 39, with minor modifications. Briefly, 50 μL of the surrogate solution was added to a 5 mL aliquot of nutrient solution. The nutrient solution samples were then extracted by HLB cartridges .