Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Summary

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

Abstract

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

Introduction

Crop plants growing in farmland may be exposed to various organic pollutants originating from anthropogenic activities^1,2. The pollutants can be taken up by plants, further causing threats to the ecosystem and human health through food chains^3,4. The xenobiotics in plants probably undergo a series of biotransformation, such as Phase I and II metabolisms⁵, generating a number of metabolites. According to the green liver concept in plants, plant metabolism can reduce the toxicity of xenobiotics^6,7. However, it has been revealed that the toxicity of some metabolites might be higher than that of their parents. For instance, the debrominated product of tetrabromobisphenol A (TBBPA) and the O-methylated product of bisphenol A (BPA) have been proven to be much more toxic than their parents^8,9, and the debromination and O-methylation comprise the main Phase I metabolism pathways in plants. Thus, the toxicity assessment solely based on pollutant parents in plants is not accurate, while the corresponding metabolites should be taken into account.

The metabolites of xenobiotics in plants are extremely complex^10,11, making it difficult to comprehensively identify and separate them. In addition, only a few standards of identified metabolites can be obtained. Hence, toxicological data of all metabolites are not available, which hinders a comprehensive toxicity assessment. This study proposed a strategy to assess the integral toxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests, providing new data for precise toxicity assessment of pollutants in plants. Our previous study has revealed that plant callus culture opens a simple and effective avenue to obtain metabolites of xenobiotics in plants¹². Accordingly, the plant callus culture was employed in this study to generate the metabolites of pollutants in plants, followed by chemical extraction and toxicological tests using a human cell line. The intestinal tract is one of the direct target organs of xenobiotics exposed to animals and humans. Caco-2 cell line has proved to be the best model for investigating the intestinal behaviors and toxicity of xenobiotics in vitro^13,14,15. Thus, the Caco-2 cell model was selected in this study.

Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production¹⁶. Their residue pollution in farmland has drawn increasing attention^17,18. Here, four commonly used triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the typical pollutants. Carrot was selected in this study as the representative plant for fresh, ready-to-eat vegetables. Carrot callus was initially exposed to the tested pesticides at a concentration of 100 mg/L. After exposure of 72 h, the metabolites were extracted to assess the cytotoxicity using Caco-2 cell line. This method can be readily extended to assess the integral cytotoxicity of metabolites of other types of pollutants in plants.

Protocol

1. Differentiation of carrot callus

NOTE: The detailed protocol for differentiation of carrot callus has been described in a previous study¹². Here is a brief description.

1. Sterilize the surface of vernalized seeds with 75% ethanol for 20 min followed by 20% H₂O₂ for 20 min. Wash the with distilled water at least 3x.
2. Sow the seeds on hormone-free agar-gelled (1% w/v) Murashige and Skoog (MS) medium (pH =5.8, autoclaved at 121 °C), and incubate them under 16 h photoperiod (350 µmol/m²s) at 26 °C for 15 days to form the seedlings.
3. Harvest the explants by cutting the hypocotyl and cotyledon of seedlings into small pieces (0.5 cm) and incubate the explants in MS medium containing auximone of 1 mg/L 2,4-dichlorophenoxyacetic acid and phytokinin of 0.5 mg/L 6-benzylaminopurine at 26 °C in the dark for 3-4 weeks to induce the callus.
4. Collect the callus (around 1 cm diameter, compact) with scalpels and forceps.

2. Treatment of carrot callus with pesticides

1. Dissolve 10 mg each of flusilazole, diniconazole, tebuconazole, and propiconazole in 100 mL of sterile MS medium with final concentrations of 100 mg/L (pH of 5.6-7.0).
   NOTE: The treatment concentration of tested pesticides was chosen as the maximum of 50% cell growth inhibition concentration (IC₅₀) to Caco-2 cell.
2. Mix 3 g of carrot callus (from step 1.4) with 10 mL of prepared pesticide solutions (from step 2.1) in glass flasks under sterile conditions.
3. Mix 3 g of carrot callus (from step 1.4) with 10 mL of aseptic MS medium in glass flasks under sterile conditions.
   NOTE: All glass flasks were autoclaved.
4. Incubate the carrot callus (from step 2.2 and 2.3) at 130 rpm and 26 °C in the dark for 72 h.
   Set all treatments in triplicate.
5. Collect the carrot callus from the medium by filtration using glass fiber filters (0.45 µm) after incubation of 72 h. Wash the callus with ultrapure water 3x.
6. Freeze-dry the callus using a freeze dryer at -55 °C, and then homogenize them using a high-throughput tissue grinder at 70 Hz for 3 min.

3. Chemical extraction of carrot callus

1. Mix 0.2 g of grounded powder of the freeze-dried callus with 3 mL of acetonitrile in a centrifuge tube (10 mL).
2. Vortex the centrifuge tube for 8 min, and then sonicate it for 5 min (150 W, 40 kHz).
3. Collect the supernatants by pipetting after centrifugation at 8,000 x g at 4 °C for 10 min.
4. Repeat the extraction procedures 3x and pool the extracts in a clean centrifuge tube (10 mL).
5. Concentrate the pooled extracts to dryness using a nitrogen blowing concentrator at 40 °C.
6. Redissolve the residues of extracts (from step 3.5) with 1 mL of Dulbecco's modified Eagle's medium (DMEM) with 0.3% dimethyl sulfoxide (DMSO).
   NOTE: The residues in step 3.6 were obtained from the extracts of pesticide-treated callus (from step 2.2).
7. Redissolve the residues of extracts (from step 3.5) with 1 mL of DMEM with 0.3% DMSO, and dissolve 1 mg of flusilazole, diniconazole, tebuconazole, and propiconazole in 4 different tubes, respectively.
   NOTE: The residues in step 3.7 were obtained from the extracts of blank callus (from step 2.3).
   NOTE: DMEM was prepared with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin).

4. Resuscitation of Caco-2 cell

NOTE: All of reagents and materials involved in Caco-2 cell tests were autoclaved for 20 min and sterilized under ultraviolet light for 2 h.

1. Remove the cryopreservation tube (storing frozen cells) from liquid nitrogen tank and quickly transfer it to a water bath at 37 °C. Continuously shake it to thaw the frozen solution.
   NOTE: Avoid water flowing over the cover of cryopreservation tube when shaking.
2. Disinfect the surface of cryopreservation tube with 75% alcohol after the frozen solution thaws. Quickly transfer the cell solution into a sterile centrifuge tube by pipetting.
3. Remove the supernatants in cryopreservation tube after centrifugation at 1,000 x g at 4 °C for 3 min. Add 1 mL of DMEM and suspend the cells by gently tapping and shaking.
4. Transfer the cell suspension (from step 4.3) into a glass flask by pipetting. Add 5 mL of DMEM in the glass flask, and gently shake the glass flask to uniformly distribute the cells.
5. Incubate the cells in an incubator at 37 °C with 5% CO₂.
   NOTE: Steps 4.2-4.4 were performed in a super clean bench.

5. Passage of Caco-2 cell

1. Remove the glass flask from the incubator and discard the culture medium when the cell density reaches over 80%. Wash the cells with PBS 3x.
2. Add 1 mL of trypsin-EDTA solution and uniformly spread it to ensure full contact with cells.
3. Remove the digestive solution when the cells shrink from adherent shape to small round point and have not suspended under the observation by microscope (100x magnification). Gently tap the flask wall to cause removal of cells.
4. Add 2 mL of DMEM, repeat rinsing of the flask wall 10x, gently tap the flask wall, and transfer 1 mL of cell suspension to another glass flask.
5. Add 5 mL of DMEM into glass flasks. Gently tap the flask wall to uniformly distribute the cells.
6. Incubate the cells in the incubator at 37 °C with 5% CO₂. Replace the culture medium with fresh DMEM every 24 h until the cell density reaches over 80%.
7. Repeat the cell passage procedures until there are enough cells (approximately 2 x 10⁶) harvested for the following exposure tests.
   NOTE: Steps 5.1-5.5 were performed in the super clean bench. The number of cells were determined by a hemocytometer¹⁹.

6. Exposure of Caco-2 cell

1. Remove the glass flasks (from step 5.7) from the incubator and repeat steps 5.1-5.4 to collect the cells.
2. Gently tap the flask wall to uniformly distribute the cells and transfer the cell suspensions to a centrifuge tube (15 mL).
3. Dilute the cell suspension (from step 6.2) with DMEM in combination with cytometry to reach a cell density of approximately 1 x 10⁵ cells/mL. Gently tap the flask wall to uniformly distribute the cells.
4. Add 100 µL of PBS in the outer wells of 96-well plate to prevent evaporation of culture medium due to the edge effect.
   NOTE: Continuously tap the wall of centrifuge tube to keep the cell suspension uniform.
5. Add 100 µL of cell suspension (from step 6.3) in the left wells of 96-well plate, and allow to rest for 10 min.
6. Incubate the cells in the incubator at 37 °C with 5% CO₂ for 48 h.
7. Remove the 96-well plate from the incubator after incubation of 48 h and discard the culture medium.
8. Set a pesticide metabolite group by adding 100 µL of solutions (from step 3.6) in each well.
   NOTE: In each well in Step 6.8, there were pesticide metabolites generated from 0.1 mg parents.
9. Set a pesticide parent group by adding 100 µL of solutions (from step 3.7) in each well for comparison.
   NOTE: In each well in Step 6.9, there were 0.1 mg pesticide parents.
10. Set a blank control by adding 100 µL of DMEM with 0.3% DMSO in each well. Use six wells for each group (step 6.8-6.9).
11. Incubate the cells in the incubator at 37 °C with 5% CO₂ for 24 h.
    NOTE: Steps 6.1-6.5 and 6.7-6.10 were performed in the super clean bench.

7. Assessment of cell viability

1. Remove the 96-well plate (from 6.11) from the incubator and discard the culture medium after exposure of 24 h. Wash the cells with PBS 2x.
2. Add 100 µL of DMEM in each well, and then add 10 µL of CCK-8 reagents. Gently shake the plate to uniformly distribute the cells.
3. Incubate the cells in the incubator at 37 °C with 5% CO₂ for 4 h.
4. Remove the 96-well plate from the incubator after incubation of 4 h and measure the optical absorbance (OD) at 450 nm by a fluorescence spectrophotometer.
5. Calculate the cell viability by the following equation:
   cell viability (%) = (OD_{experimental group} - OD_DMEM) / (OD_control - OD_DMEM)×100%.
   NOTE: Steps 7.1-7.2 were performed in the super clean bench.

Results

Figure 1 represents the schematic of proposed method for generation, extraction, and cytotoxicity assessment of pesticide metabolites in carrot callus. In Figure 2, the uptake and metabolism kinetics curves of tested pesticides, from which we can find that the concentrations of pesticides in culture media were exponentially decreased, while those in carrot callus began to increase, peaking at 4 or 8 h, followed by a gradual decrease. These results suggested that...

Discussion

This protocol was developed to assess the integral cytotoxicity of metabolites of triazole pesticides in plants by combining plant callus and human cell models. The critical steps for this proposed protocol are the culture of plant callus and Caco-2 cell. The most difficult part and relative advice for plant callus culture have been provided in our previous study¹². Here, it should be noted that cell maintenance is the most difficult part for Caco-2 cell culture, because the cells are easily infec...

Materials

Name	Company	Catalog Number	Comments
2,4-dichlorophenoxyacetic acid	WAKO	1 mg/L
20% H2O2	Sinopharm Chemical Reagent Co., Ltd.	10011218-500ML
6-benzylaminopurine	WAKO	0.5 mg/L
75% ethanol	Sinopharm Chemical Reagent Co., Ltd.	1269101-500 mL
96-well plate	Thermo Fisher
Acetonitrile	Sigma-Aldrich
Artificial climate incubator	Ningbo DongNan Lab Equipment Co.,Ltd	RDN-1000A-4
Autoclaves	STIK	MJ-Series
Caco-2 cells	Nuoyang Biotechnology Co.,Ltd.
CCK8 reagents	Nanjing Jiancheng Bioengineering Institute, China	G021-1-3
Centrifuge	Thermo Fisher
CO2 incubator	Labtrip	HWJ-3-160
Dimethyl sulfoxide	Solarbio Life Sciences	D8371
Diniconazole, 98.7%	J&K Scientific	83657-24-3
Dulbecco's modified Eagle's medium	Solarbio Life Sciences	11965-500 mL
electronic balance	Shanghai Precision Instrument Co., Ltd	FA1004B
Fetal bovine serum	Cellmax
Fluorescence spectrophotometer	Tecan	Infinite M200
Flusilazole, 98.5%	J&K Scientific	85509-19-9
Freeze dryer	SCIENTZ
High-throughput tissue grinder	SCIENTZ
Inverted microscope	Leica Biosystems	DMi1
Milli-Q system	Millipore	MS1922801-4L
Murashige & Skoog medium	HOPEBIO	HB8469-7
Nitrogen blowing concentrator	AOSHENG	MD200-2
PBS	Solarbio Life Sciences	P1022-500 mL
Penicillin-Streptomycin Liquid	Solarbio Life Sciences	P1400-100 mL
Propiconazole, 100%	J&K Scientific	60207-90-1
Research plus	Eppendorf	10-1000 μL
Seeds of Little Finger carrot (Daucus carota var. sativus)	Shouguang Seed Industry Co., Ltd
Shaking Incubators	Shanghai bluepard instruments Co.,Ltd.	THZ-98AB
Tebuconazole, 100%	J&K Scientific	107534-96-3
Trypsin-EDTA solution	Solarbio Life Sciences	T1300-100 mL
Ultrasound machine	ZKI	UC-6
UV-sterilized super clean bench	AIRTECH
Vortex instrument	Wuxi Laipu Instrument Equipment Co., Ltd	BV-1010