Article pubs.acs.org/JAFC
Uptake and Accumulation of Nephrotoxic and Carcinogenic Aristolochic Acids in Food Crops Grown in Aristolochia clematitis-Contaminated Soil and Water Weiwei Li, Qin Hu, and Wan Chan* Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China S Supporting Information *
ABSTRACT: Emerging evidence has suggested aristolochic acids (AAs) are linked to the development of Balkan endemic nephropathy (BEN), a chronic renal disease affecting numerous farmers living in the Balkan peninsula. However, the pathway by which AAs enter the human food chain and cause kidney disease remains poorly understood. Using our previously developed analytical method with high sensitivity and selectivity (Chan, W.; Lee, K. C.; Liu, N.; Cai, Z. J. Chromatogr. A 2007, 1164, 113− 119), we quantified AAs in lettuce, tomato, and spring onion grown in AA-contaminated soil and culture medium. Our study revealed that AAs were being taken up from the soil and bioaccumulated in food crops in a time- and dose-dependent manner. To the best of our knowledge, this study is the first to identify one of the possible pathways by which AAs enter our food chain to cause chronic food poisoning. Results also demonstrated that AAs were resistant to the microbial activity of the soil/water. KEYWORDS: aristolochic acids, Balkan endemic nephropathy, uptake, lettuce, high-performance liquid chromatography
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INTRODUCTION Balkan endemic nephropathy (BEN) is a chronic kidney disease affecting numerous farmers living along the Danube River.1 Cases of BEN were reported in rural regions of Bosnia, Bulgaria, Croatia, Romania, and Serbia. Despite significant research conducted over the past decades, the etiology of BEN is still not fully understood.1−3 Evidence suggests that chronic food poisoning by mycotoxic ochratoxin A, polycyclic aromatic hydrocarbons, heavy metals, and radioactive materials may be associated with disease development.1−3 However, the pathophysiology of BEN remains controversial and requires further investigations. Aristolochia-derived aristolochic acids (AAs, Figure 1) have recently been discovered to contribute to the development of kidney fibrosis and cancer in the urinary tracts of BEN patients.4−8 Specific evidence included the identification of AAinduced DNA adducts in the internal organs of BEN patients.4,5 Furthermore, farmlands in Romania were found to be heavily contaminated with Aristolochia clematitis.6,9 Therefore, scientists hypothesized that the AA-containing fruit of A. clematitis were being harvested together with the wheat and contaminated the food ingredient of farmers living in the endemic villages.10 The prolonged intake of AA-contaminated food is suspected to be responsible for the development of BEN. However, the pathways by which AAs enter our food chain remain controversial, and the link between chronic dietary poisoning by AAs and BEN has not yet been thoroughly established. With our prior knowledge that environmental pollutants are taken up by plants,11−16 the goal of this study was to test the hypothesis that AAs were translocated and bioaccumulated in food crops to cause chronic dietary poisoning. Emerging evidence has suggested AAs could be taken up and accumulated in food crops. For example, AAs were detected in the soil samples collected from both the BEN and non-BEN areas, and © 2015 American Chemical Society
Figure 1. Chemical structure of aristolochic acids (AAI, R = OCH3; AAII, R = H) and Zn/H+-induced nitroreduction converting the nonfluorescing AAs to aristolactams (aristolactam I, R = OCH3; aristolactam II, R = H) that strongly fluoresce for HPLC-FLD analysis.
AAs were observed to have been absorbed into the roots of maize and cucumber.6 However, the root tissues of maize and cucumber from which AAs were detected are not consumed directly by humans, and exposures carried out in a nutrient solution do not simulate realistic environmental exposure. Therefore, a more direct relationship between plant contamination by AAs and chronic food poisoning has been more rigorously established. Received: Revised: Accepted: Published: 107
October 20, 2015 December 9, 2015 December 10, 2015 December 10, 2015 DOI: 10.1021/acs.jafc.5b05089 J. Agric. Food Chem. 2016, 64, 107−112
Article
Journal of Agricultural and Food Chemistry
harvested at days 7 and 14 after the start of treatment, processed, and analyzed by HPLC-FLD as described below. The samples including the fruit of tomato and the bulbs and leaves of spring onion collected before the treatment were used as control. Cultivation of Lettuce in AA-Contaminated Water. To investigate the uptake and bioaccumulation of AAs in food crops, butterhead lettuce was also grown in AA-spiked culture medium. The concentrations of AAs in both the lettuce leaves and the culture medium were monitored on a weekly basis using the HPLC-FLD method described below. Control experiments were conducted by cultivating lettuce in nonspiked culture medium as well as by analyzing AA-fortified medium with no lettuce growing on it. Sample Preparation. Extraction. After three washings with Milli-Q water, lettuce leaves were air-dried and cut into small pieces. Approximately 400 mg of the sliced samples was accurately weighed and extracted with 4 mL of the extraction solvent (methanol/water/ acetic acid, 70:25:5; v/v). After sonication at 50 °C for 1 h, the samples were reduced by Zn/H+ and cleaned by solid phase extraction (SPE) before being analyzed by HPLC-FLD. Zn/H+ Treatment. By using a previously described method, AAs were converted to their corresponding aristolactams for their sensitive detection by HPLC-FLD. Approximately 1 mL of the sample extract was transferred to a 1.5 mL polystyrene Eppendorf tube containing 10 mg of zinc powder. After reacting at room temperature for 15 min, the sample was centrifuged at 14000 rcf for 5 min, and the supernatant was diluted with water twice before the SPE cleanup. SPE Purification. A 1 mL sample extract was loaded onto a C18 SPE column (Waters, Milford, MA, USA) that was conditioned sequentially with 5 mL of methanol and 5 mL of water. After washing with 1 mL of water and 1 mL of water/methanol (50:50, v/v), the columns were eluted with 1 mL of methanol. The eluates were collected and evaporated to dryness under nitrogen. The residue was dissolved in 100 μL of methanol and centrifuged at 14000 rcf for 5 min prior to HPLC-FLD analysis. HPLC-FLD Analysis. HPLC-FLD analysis was performed on an Agilent 1200 HPLC system coupled with a fluorescence detector (Palo Alto, CA, USA). Sample extract (10 μL) was injected onto a Waters XSelect C18 column (3.0 mm × 100 mm, 2.5 μm) with a constant temperature at 50 °C. The column was eluted with a binary solvent system consisting of water (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. The solvent gradient started from 20% B, was programmed to 100% in 15 min, and was held for 5 min before reconditioning. The excitation and emission wavelengths of the fluorescence detector were set at 393 and 470 nm, respectively. Data Analysis. HPLC-FLD analysis of the derivatized sample extracts revealed the total aristolactam level (Ct) from both the nitroreduction of AA and native aristolactams (Ci) in the leaves (Figure 2).17 The concentration of AA was determined by subtracting Ci from Ct. Thus, the HPLC-FLD analysis comprised two parts: (1) analysis of the underivatized sample extract to determine Ci and (2) analysis of the derivatized sample extract to determine Ct.
With the goal of building a better connection between environmental pollution by AAs and BEN, we investigated in this work the root uptake and transfer into the leaf of lettuce, the fruit of tomato, and the bulb and leaf of spring onion grown in AA-contaminated soil. By using our previously developed high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method of high sensitivity and selectivity (Figure 1),17 we report the first identification of AAs in the edible parts of plants grown in AA-contaminated soil. This method uses the Zn/H+-induced nitroreduction of AAs to convert AAs into strongly fluorescing aristolactams and to facilitate the precise determination of AAs by HPLC-FLD. The identification of AAs in lettuce leaves was further validated using UPLC-MS analysis.
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MATERIALS AND METHODS
Caution. AAs are human carcinogens and should be handled with care. Chemicals. All chemicals and reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise noted. Aristolochic acids (AAs; 1:1 mixture of AAI and AAII, 96% purity) were purchased from Acros (Morris Plains, NJ, USA). Dried A. clematitis fruit was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The lettuce seeds were purchased from Mr. Fothergill’s (Newmarket, UK). HPLC grade acetonitrile and methanol were purchased from Tedia (Fairfield, OH, USA). Deionized water was further purified by a Milli-Q Ultrapure water purification system (18.2 MΩ, Millipore, Billerica, MA, USA) before use. Preparation of Herbal Extracts. Dried A. clematitis fruits were homogenized in a household blender before use. Approximately 20.1 g of the powder was accurately weighed and extracted by using 1 L of distilled water by sonication for 1 h at 40 °C. The herbal extracts after filtering through a 20 μm filter were analyzed by HPLC and stored at 4 °C until used. Stability of AAs in Soil. The soil was randomly obtained around the campus of the Hong Kong University of Science and Technology and repeatedly mixed until the final soil material was as homogeneous as possible. The stability of AAs toward soil microbial activities of soil was investigated by spiking 5 g of soil with 100 μL of herbal extracts containing 14.58 μg/mL of AAs (11.72 μg/mL AAI, 2.86 μg/mL AAII) in a 50 mL centrifuge tube. After mixing by inversion, 1 g of the soil sample was sampled at days 0, 7, 14, 21, and 28 days after spiking. The soil samples were then extracted using 3 mL of extraction solvent (methanol/water/acetic acid, 70:25:5; v/v), derivatized by Zn/H+ treatment, SPE enriched, and analyzed by HPLC-FLD as described below. Cultivation of Lettuce in AA-Contaminated Soil. To mimic the natural agricultural conditions in the AA-contaminated land in the Balkan peninsula, Lactuca sativa (red oak and butterhead lettuce) after germination were inoculated in pots and allowed to grow in the ambient environment with regular watering. After growing to ∼10 cm in diameter, the red oak lettuces were divided into three groups (n = 3) and treated with 15 mL of aqueous herbal extracts daily for three consecutive weeks. Plants in the high-dosage group received aqueous extracts containing 14.58 μg/mL AAs (11.72 μg/mL AAI, 2.86 μg/mL AAII), whereas the low-dosage group received herbal extracts containing 3.64 μg/mL AAs (2.92 μg/mL AAI, 0.72 μg/mL AAII). Lettuce receiving an equal volume of water was used as the control group. Similarly, butterhead lettuce were also cultivated and treated with herbal extracts containing 7.3 μg/mL AAs (5.87 μg/mL AAI and 1.43 μg/mL AAII). The lettuce leaves were sampled weekly at days 0, 7, 14, 21, 25, 28, 35, 42, 49, and 56 days, whereas soils were sampled at day 60 after the start of irrigation. The samples were then processed immediately for HPLC-FLD analysis. Cultivation of Tomato and Spring Onion in AA-Contaminated Soil. Using a similar approach, tomato plants (n = 3) after fruiting and spring onion (n = 3) were irrigated daily with 15 mL of aqueous herbal extracts containing 14.58 μg/mL of AAs for 2 weeks. The fruits of tomato and the whole plant of spring onion were
Figure 2. Schematic illustration of the sample preparation, HPLCFLD, and data analysis procedures for the determination of AAs and aristolactams. 108
DOI: 10.1021/acs.jafc.5b05089 J. Agric. Food Chem. 2016, 64, 107−112
Article
Journal of Agricultural and Food Chemistry Consequently, the molar concentrations of AAs in the samples were measured by subtracting Ci from Ct. Calibration and Method Validation. To achieve linearity in the HPLC-FLD method, a group of the AAs at five concentration levels was prepared by continuous dilution of the stock solution with blank sample matrix. The concentrations of AAI and AAII varied from 0.015 to 7.331 μM and from 0.016 to 8.039 μM, respectively (Table 1).
The highest analytical errors were 9.3 and 7.5% for AAI and AAII, respectively. Depicted in Table 1 are the calibration slopes, intercepts, and correlation coefficients (R2) for the determination of AAI and AAII. The data of accuracy and precision shown in Tables 1 and 2 reveal good method performance of the Table 2. Intra- and Interday Precision and Accuracy of the Developed HPLC-FLD Method for the Determination of AAI and AAII in Lettuce
Table 1. Linear Regression Parameters of the Calibration Curves and Limits of Detection of the Developed HPLCFLD Method for the Determination of AAs in Lettuce linear range (μM) slope intercept R2 LOD (ng/mL) LODa (ng/g) a
AAI
AAII
0.015−7.331 652.56 −5.6429 0.9996 0.73 0.73
0.016−8.039 528.92 −0.2668 0.9999 0.92 0.92
precision
The LOD is the sample extract based on 400 mg of lettuce sample. a
The calibration curves were established by plotting the averaged peak areas of aristolactams (n = 3) versus the AAs concentration in the calibration standards. The limit of detection (LOD) was established as the amount of analyte in the blank sample extract that generated a signal that is 3 times the signal-to-noise ratio.18,19 The instrumental precision was evaluated through analysis of blank lettuce extracts spiked with AAs at three concentrations (AAI, 0.0126, 0.126, and 1.603 μg/g; AAII, 0.0124, 0.124, and 1.617 μg/g) on the same day (n = 7) and over individual days in 3 weeks (n = 7). The accuracy of the method was determined by spiking AA at the three stated concentrations to blank sample matrix, derivatized, and analyzed using the above-mentioned method. UPLC-MS Analysis. UPLC-MS and MS/MS analyses of the aristolactam-containing fractions were performed on an Acquity HPLC system coupled with a Xevo G2 Q-TOF mass spectrometer with a standard electrospray ionization interface (Waters, Milford, MA, USA). A Waters BEH C18 column (2.1 mm × 100 mm, 1.8 μm) was used to separate the aristolactams from the sample matrix. The mobile phases were 0.1% formic acid (A) and methanol (B). The mass spectrometer was operated in positive electrospray ionization mode with the desolvation gas temperature and capillary voltage set at 400 °C and 3.0 kV, respectively.
accuracy
concn added (μg/g)
intradaya (% RSD)
interdaya (% RSD)
concn foundb (μg/g)
error (%)
AAI
0.0126 0.126 1.603
7.9 6.0 2.7
9.4 10.4 3.9
0.014 ± 0.01 0.1201 ± 0.04 1.731 ± 0.15
9.25 4.72 7.97
AAII
0.0124 0.124 1.617
4.1 7.1 5.5
6.0 8.0 6.6
0.012 ± 0.01 0.12 ± 0.03 1.50 ± 0.29
6.58 3.91 7.54
n = 7. bn = 3.
developed HPLC-FLD method for the determination of AAI and AAII in lettuce. Stability of AAs toward Soil Microbial Activities. To investigate the chemical and in vivo stability of AAs upon soil microbial activities, the levels of AAs in the AA-fortified soil were monitored weekly by HPLC-FLD for a period of 1 month. Our results from direct analysis of the sample extract showed no detectable amount of aristolactam (Ci, Figure 2), which is the major metabolite of AA,20 in the soil samples. Results also showed no significant difference in AAs concentrations (Ct − Ci) in the soil samples collected within the 1 month period (Figure 3). These results indicated that AAs were resistant to soil
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RESULTS AND DISCUSSION Linearity, Detection limit, Precision, and Accuracy of the HPLC-FLD Method. Calibration curves for AAI and AAII were established by derivatizing and HPLC-FLD analyzing AAs standard solution mixtures using the optimized method described above, with the concentrations of AAI and AAII varied from 0.015 to 7.331 μM and from 0.016 to 8.039 μM, respectively. The peak areas of aristolactams increased linearly over the AAs concentrations, with coefficients of determination (R2) >0.9996, indicating that the analysis method combining Zn/H+-induced nitroreduction and that HPLC-FLD analysis was highly quantitative. Under the optimized HPLC-FLD conditions, detection limits of 0.73 and 0.92 ng/mL were achieved for AAI and AAII, respectively. These detection limits correspond to method detection limits of 0.73 and 0.92 ng of AAI and AAII per gram of lettuce, respectively, when 400 mg of lettuces was processed and analyzed as described under Materials and Methods. For both AAI and AAII, the intraday precisions of the analysis at the three stated levels were of relative standard deviation
