Uptake, subcellular distribution and metabolism of ¹⁴C-caffeine in leafy vegetables from water

Highlights

• ¹⁴C-labeling analysis and LC-QTOF-MS were used to investigate caffeine metabolism in plants.

• Caffeine and metabolites were primarily presented in the solution fraction of leaves.

• Caffeine and metabolites were not homogeneously distributed in vegetable leaves.

• Caffeine in vegetables underwent demethylation and mineralization to release CO₂.

Abstract

Irrigation with treated wastewater could lead to the accumulation of caffeine in agricultural fresh. Caffeine is one of the most frequently detected compounds in treated wastewater; however, little is known about its subcellular distribution and metabolism in vegetables. This study reported the uptake, subcellular distribution, and metabolism of ¹⁴C-caffeine in Chinese flowering cabbage and water spinach. The results showed that 98% of caffeine lost from solution after 768 h of cultivation. Caffeine was taken up by vegetables and most ¹⁴C-activity was accumulated in the bottom leaves. At the subcellular level, ¹⁴C-activity was mainly distributed in the organelles in root and stem cells, while in the leafy cells it was dominant in the solution. The metabolism of caffeine was investigated using LC-QTOF-MS. Caffeine underwent demethylation forming xanthine and theobromine, and mineralization to release CO₂. Approximately 40.2% of the initially applied caffeine was accumulated in Chinese flowering cabbage as the parent compound (28.3%) and metabolites (11.9%), and 50.9% of the added caffeine was mineralized to CO₂ after 768 h of exposure. The knowledge obtained herein is key to evaluating potential risks of caffeine present in treated wastewater, and the quality and safety of agricultural fresh produced by irrigation with treated wastewater.

Introduction

Plant uptake and accumulation of chemicals of emerging concern such as many pharmaceuticals and personal care products (PPCPs) pose potential risks to human health (Ebele et al., 2017). PPCPs include many diverse and commonly used chemicals that have been frequently detected in various environmental matrices, such as in soils, surface and groundwater (Spongberg et al., 2011). Many PPCPs can survive wastewater treatment processes, enter the effluents (treated wastewater) and be disseminated to the surrounding water bodies. Agricultural irrigation with treated wastewater or contaminated surface water can transfer these PPCPs to the edible parts of fresh produce (Kot-Wasik et al., 2007, Wu et al., 2013). Because of water scarcity in many arid and semi-arid regions, treated wastewater has been increasingly used in agricultural irrigation. Approximately 5.6 billion tons of treated wastewater is used for agricultural irrigation per year globally, and more than 85% of treated wastewater is used for irrigation in Israel (Ben Mordechay et al., 2018, Fu et al., 2019). This practice could lead to more accumulation of PPCPs in agricultural produce. The consumption of these PPCP-laden food products could elevate the risks associated with human health (Wu et al., 2015, Schapira et al., 2020).

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid, an active constituent in many drinks such as coffee and tea, and is the most common psychostimulant administered to humans. Caffeine has been also used as a model chemical to track the wastewater sources (Cantwell et al., 2018), and to distinguish human from animal pollution (Glassmeyer et al., 2005, Gourmelon et al., 2010). Caffeine, as one of the most frequently detected PPCPs in the environment, has been found at relatively high concentration in the effluents from wastewater treatment plants (Spongberg et al., 2011, Hedgespeth et al., 2012). The concentration of caffeine was found at μg L⁻¹ levels in the influents to wastewater treatment plants in American, Asian and European countries (Nakada et al., 2017, Paíga et al., 2019). Spongberg et al. (2011) found that caffeine could reach 1.1 mg L⁻¹ in Costa Rican surface water originating from the nearby coffee bean processing facilities. Caffeine was detected at 100% of frequency in the surface water and sediments of the Guanting Reservoir and its upstream, Hebei Province, China; the average concentration was 208 and 338 ng L⁻¹ in the reservoir and upstream, and 1430 and 1020 ng g⁻¹ in the associated sediments (Zhang et al., 2018).

Caffeine can be metabolized in plants, and their intermediate or end products still contain the bioactive moieties (Chuang et al., 2018). Most previous research efforts have been dedicated to investigate caffeine degradation and metabolism in tea or coffee trees (Mazzafera, 2004, Wang et al., 2016, Zhou et al., 2020). Theophylline and theobromine are the main metabolites in caffeine-rich plants such as coffee and tea, resulting from the demethylation at the purinic ring of caffeine (Wang et al., 2016, Zhou et al., 2020). It is believed that caffeine yields metabolites theophylline and trimethyluretic acid by cytochrome P450s at a high concentration, while yielding paraxanthine at a low concentration (Jandova et al., 2019, Tian et al., 2019). Pierattini et al. (2016) reported that exogenous caffeine in Populus alba was metabolized into theobromine and theophylline. Chuang et al. (2018) found that caffeine underwent major demethylation and oxidation reactions in lettuce with 54% of caffeine metabolized within six days. However, it is still unclear about the whole metabolism processes of caffeine and metabolites in vegetables. Moreover, no information is available on the subcellular distribution and mineralization of caffeine in plants. Therefore, a systematic investigation using the mass conservation of ¹⁴C-caffeine and mass spectroscopy analysis was conducted to shed light on the distribution of caffeine and metabolites at subcellular levels and the whole metabolism process in selected vegetables.

The objective of this research is to systematically evaluate the uptake, subcellular distribution, metabolism and mineralization of caffeine in Chinese flowering cabbage and water spinach using ¹⁴C-labeled caffeine. Chinese flowering cabbage and water spinach are two types of commonly consumed fresh vegetables. The combination of ¹⁴C-labeling method and liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer (LC-QTOF-MS) analysis facilitated the track of dynamic changes of ¹⁴C-caffeine and metabolites in plants, and further visualization of the distribution of ¹⁴C-caffeine and metabolites in plants using the radioautography technique (Fig. S1). The results from this study provide new insights into uptake, distribution and metabolism of caffeine in vegetables. To the best of our knowledge, for the first time we demonstrated the accumulated caffeine in vegetables could be mineralized to produce CO₂, which represents a new decomposition pathway for the PPCPs accumulated in plants. The results from this study are informative to the beneficial use of treated wastewater in agricultural irrigation, and the evaluation of potential risks associated with human exposure via dietary consumption of PPCP-laden agricultural produce.