|Year : 2021 | Volume
| Issue : 2 | Page : 240-245
Determination of the bioaccessibility of cadmium in golden thread by physiologically based extraction test digestion using the in vitro/Caco2 cell model and subsequent risk assessment
Tian-Tian Zuo1, Fei-Ya Luo2, Hong-Yu Jin2, Shu-Xia Xing2, Bo Li2, Kun-Zi Yu1, Shuai Kang3, Lei Sun1, Shuang-Cheng Ma2
1 Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing; School of Pharmacy, Xi'an Jiaotong University, Xi'an, China
2 Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing, China
3 Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing; College of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang, China
|Date of Submission||22-May-2020|
|Date of Acceptance||28-Aug-2020|
|Date of Web Publication||24-Mar-2021|
Dr. Lei Sun
National Institutes for Food and Drug Control, No. 2 Tiantan Xili, Dongcheng District, Beijing 100050
Prof. Shuang-Cheng Ma
National Institutes for Food and Drug Control, No. 2 Tiantan Xili, Dongcheng District, Beijing 100050
Source of Support: None, Conflict of Interest: None
Background: The ingestion of golden thread contaminated with heavy metals through the food chain leads to detrimental effects to human health. During digestion, not all of the heavy metals could be released to the gastrointestinal tract and readily to be absorbed by human body. Thus, bioaccessibility is an important issue in health risk assessments. Aims and Objectives: The aims and objectives of this study were to investigate the bioaccessibility of Cd in golden thread and assess the associated health risks based on the exposure to bioaccessible Cd. Materials and Methods: Inductively coupled plasma mass spectrometry (ICP-MS) has been applied to determine the Cd content in golden thread. Physiologically based extraction test (PBET) digestion was performed in the in vitro/Caco2 cell model to investigate the bioaccessibility of Cd in golden thread. Furthermore, the target hazard quotient (THQ) was used to assess the risks of the total and the bioaccessible content of Cd in golden thread. Results: The results revealed that the total Cd content in six batches of golden thread ranged from 3.203 to 5.723 mg/kg. After uptake by Caco2 cells, the bioaccessibility of Cd ranged from 42.36% to 59.73 %. The results of the risk assessment indicated that prior to uptake by Caco2 cells, the THQ values of Cd for all batches of golden thread were greater than 1. However, after uptake by Caco2 cells, the THQ values of Cd in all samples were less than 1, thus suggesting that the risks were at a safe level. Conclusion: This study was the first to perform health risk assessment with bioaccessible heavy metals present in traditional Chinese medicine by PBET digestion using an in vitro/Caco2 cell model, thus enabling us to obtain more accurate and objective results while allowing us to avoid unnecessary government intervention and to establish more reasonable limit standards for heavy metals.
Keywords: Bioaccessibility, cadmium, golden thread, physiologically based extraction test digestion in vitro/Caco2 cell model, risk assessment
|How to cite this article:|
Zuo TT, Luo FY, Jin HY, Xing SX, Li B, Yu KZ, Kang S, Sun L, Ma SC. Determination of the bioaccessibility of cadmium in golden thread by physiologically based extraction test digestion using the in vitro/Caco2 cell model and subsequent risk assessment. World J Tradit Chin Med 2021;7:240-5
|How to cite this URL:|
Zuo TT, Luo FY, Jin HY, Xing SX, Li B, Yu KZ, Kang S, Sun L, Ma SC. Determination of the bioaccessibility of cadmium in golden thread by physiologically based extraction test digestion using the in vitro/Caco2 cell model and subsequent risk assessment. World J Tradit Chin Med [serial online] 2021 [cited 2021 Dec 3];7:240-5. Available from: https://www.wjtcm.net/text.asp?2021/7/2/240/316611
| Introduction|| |
Golden thread (Coptis chinensis Franch.) is a commonly used traditional Chinese medicine (TCM) that was first described in “Shen Nong's Materia Medica” during the Eastern Han Dynasty of China. It has been identified to possess excellent pharmacological activities that include detoxifying, anti-infection, anti-jaundice, anti-inflammatory, and anti-tumor effects., However, the golden thread may be polluted by heavy metals during cultivation, harvesting, and processing. Cadmium (Cd) is a heavy metal that represents a global concern due to its lack of biodegradability in the environment and its known health risks to humans. Studies have demonstrated that heavy metals exert toxic effects by impairing cell, protein, and DNA functions. Chronic exposure to Cd can cause a decline in cognitive capacity, reproductive deficiencies, fractures, and diabetes.,,, It has been reported that the Cd content in the golden thread is of concern. The ingestion of golden thread contaminated with Cd through the food chain leads to the accumulation of this harmful contaminant in living organisms, and this may pose potential risks to public health.
Previous studies examining the risks associated with food, water, and soil contaminated by Cd have been performed.,,,, In general, the majority of the studies focused on the total content of Cd, which provided meaningful information regarding the overall contamination levels. However, such determinations cannot reflect the actual levels of heavy metals that accumulate within the human body, and this may result in an overestimation of the exposure levels and the potential health risks. Therefore, a more accurate understanding of the bioavailability of Cd is crucial to assess its health risk to humans. This understanding will be achieved through investigations examining the oral bioaccessibility of Cd in golden thread and by developing an improved risk assessment model of the contaminant based on bioaccessibility to guide the clinical use of golden thread.
Bioaccessibility is defined as the fraction of Cd that can be released from the matrix into the gastrointestinal tract and then readily absorbed by the human body., Comparatively, bioaccessibility enables accurate measurement of the oral exposure risk of Cd in golden thread. Recently, various in vitro digestion models have been proposed and have been used to determine Cd bioaccessibility based on their considerable advantages that include simplicity, rapidity, energy savings, low cost, and ease of control. Moreover, these models allow for improved reproducibility and can thus provide a cost-effective estimate of in vivo conditions. The physiologically based extraction test (PBET) proposed by Ruby in 1996 has been one of the most pioneering and broadly accepted in vitro models, and this test has been successfully used to assess the bioaccessibility of a number of heavy metals. Furthermore, the Caco-2 cell line that is derived from colon carcinoma has been widely used as an in vitro model for mimicking the absorption process of the human intestinal epithelium barrier to determine the bioaccessibility of various substances.,
To the best of our knowledge, few studies have assessed the bioaccessibility of Cd derived from golden thread using in vitro digestion/Caco2 cells. Given the widespread use of golden thread in China and the associated concerns regarding human health, the goals of this study include (1) determining the total Cd levels in the golden thread, (2) investigating the bioaccessible levels and bioaccessibility of Cd in the golden thread after simulating human in vitro digestion using a PBET/Caco-2 cell model, and (3) assessing the associated health risks based on the exposure to bioaccessible Cd.
| Materials and Methods|| |
Reagents and samples
Double-deionized water was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). The Cd standard solution was obtained from the National Standard Material Research Center (Beijing, China). Tuning solutions containing Li, Y, Ce, Tl, and Co (Part# 5185–5959) and an internal standard solution containing 6Li, Sc, Ge, Rh, In, Tb, Lu, and Bi (Part# 5188–6525) were purchased from Agilent Technologies (Folsom, CA, USA). Supra-pure trace metal-grade concentrated nitric acid (HNO3, 65.0%) was purchased from Merck (Merck, Munchen, Germany). Unless otherwise stated, chemicals for in vitro PBET digestion were purchased from Sigma Chemical (St. Louis, MO, USA). For the Caco-2 cell model, fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM) containing glucose, and 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco (Carlsbad, CA, USA). A total of six batches of golden thread were collected from TCM markets and retail pharmacies in the provinces of Sichuan [Table 1]. The samples were authenticated by Dr. Kunzi Yu. Voucher specimens were deposited in the National Institutes for Food and Drug Control, Beijing, China.
Analysis of the total cadmium concentrations by inductively coupled plasma mass spectrometry
The samples were ground into a homogenous powder, and 0.5 g of this powder was placed into microwave digestion tubes (CEM Corporation, Matthews, NC, USA) for digestion with 8.0 mL HNO3. After digestion, the acid in the vessels was dispelled for 40.0 min. The digestion solution was then diluted to 50.0 mL with Milli-Q water. Analysis of the Cd contents was performed using an Agilent 7700X inductively coupled plasma mass spectrometry (ICP-MS) (Agilent Technologies, Folsom, CA, USA) based on our previously published methods. The instrumental conditions for ICP-MS included RF power of 1550 W, a plasma gas flow rate of 15.0 L/min, a peristaltic pump rate of 0.2 r/s, an atomizing chamber temperature of 2.0°C a carrier gas flow rate of 0.8 L/min, a He gas flow rate of 5.0 mL/min, an auxiliary gas flow rate of 0.8 L/min, a sampling depth of 10.0 mm, and 100 sweeps.
In vitro digestion
The bioaccessibility of Cd in golden thread samples was investigated using the PBET method that involved two extraction steps that included a gastric and an intestinal phase. Briefly, during the gastric extraction stage, 0.50 g of the sieved samples were mixed with 50 mL of a simulated gastric solution (1.25 g of pepsin, 0.50 g of sodium citrate, 500 μL of acetic acid, 0.50 g of sodium malate, and 420 μL of lactic acid were brought to a total volume of 1 L with deionized water) in a 50 mL polypropylene centrifuge tube. The pH of the gastric solution was adjusted to 2.0 by the addition of HCl. Then, all the tubes were transferred to an 80 rpm shaking incubator at 37°C for 1 h. The solution was centrifuged at 3500 ×g for 5 min, and a total of 25 mL of the supernatant was collected. Further, the supernatant was concentrated to approximately 3 mL using an electro-thermal plate. After cooling, 5 mL of nitric acid was added to the concentrated supernatant that was ready for digestion. After digestion, deionized water was added to the digested solution to a volume of 50 mL to simulate the gastric fraction for further determination.
In the intestinal stage, the residue from the gastric stage was added to 50 mL of a simulated intestinal solution containing bile salts and pancreatin (pH 7.0). Then, the solution was incubated at 37°C and shaken at 80 rpm for 4 h. The solution was centrifuged at 3500 ×g for 5 min, and a total of 25 mL of the supernatant was collected and then concentrated to 3 mL using an electro-thermal plate. The supernatant was then mixed with 5 mL of nitric acid for digestion. Finally, deionized water was added to the digested solution to a volume of 50 mL to simulate the intestinal fraction for further measurement.S
Caco-2 cell model
Human colon adenocarcinoma Caco-2 cells were purchased from the Shanghai Institutes for Biological Science of the Chinese Academy of Medical Sciences (Shanghai, China). Caco-2 cells were maintained in DMEM supplemented with 10% (v/v) FBS and 2.3 g/L sodium bicarbonate. Cells were incubated in a humidified incubator containing 5% CO2 at 37°C. The culture medium was changed every 2 d, and the cells were transferred using 0.25% trypsin/EDTA when the cells were 80% confluent.
Cell differentiation and uptake experiments were performed in a two-chamber well using polyester membranes in 24-well transwell plates (12 mm diameter, pore size 0.4 μm; Millipore, Milford, MA, USA). In this system, Caco-2 cells were maintained on a porous support that divided the well into two compartments that were defined as apical and basolateral. Caco-2 cells were seeded at 5.0 × 104 cells/mL. To achieve mature Caco-2 cells and differentiated confluent cell monolayers, the cells were incubated for 21 days, and the culture medium was changed every 2 days during incubation. During the period of growth and differentiation, the integrity of the cell monolayer was monitored by measuring the transepithelial electrical resistance (TEER) using a Millicell ERS (WPI Corporation, USA). Only cell models possessing a TEER >200 s937;/cm2 were used for further testing.
Further experiments were performed at 21 d postseeding. A volume of 0.5 mL of the intestinal fraction was added to the apical side, and a 0.5 mL volume of FBS-free DMEM was added to the basolateral side. After 4 h of incubation at 37°C and 5% CO2, the medium from the basolateral compartments was harvested and filtered through a 0.22 μm membrane (Millipore, USA) for further analysis using ICP-MS. Control groups were prepared for each test. The bioaccessibility of Cd in both the gastric and the intestinal phases after uptake by Caco2 cells was calculated as follows:
Health risk assessment
To assess the health risks due to chronic exposure to Cd via consumption of golden thread, the target hazard quotient (THQ) was calculated using the following formula:
Where THQ is the THQ of Cd in the golden thread. EF is the exposure frequency, which is 90 days/year based on our previous studies. Ed is the exposure duration, which is 20 years. IR is the ingestion rate of TCM. According to the statistical data from our previous study, the mean value of IR was 200 g/day. C is the total Cd content in the golden thread samples (mg/kg). SF is the safety factor, which is 10 for TCM. t represents the transfer rate of Cd from herbal medicinal materials to decoctions or preparations (%), and this rate is 14%. For the daily intake of total Cd content, BA is 1, and for the daily intake of bioaccessible Cd, BA is the bioaccessibility of Cd calculated by the formula (1). W is the average body weight (60 kg). AT is the average time of exposure to the golden thread, which is 365 days/year × 70 years. RfD is the oral reference dose suggested by USEPA for Cd, which is 1 μg/kg bw/day. If THQ is <1, the exposure to the golden thread is associated with acceptable risk. Otherwise, the health risk of the exposed population may be of concern.
| Results and Discussion|| |
The quantitative analysis of Cd in golden thread samples was performed using a calibration curve obtained by diluting a standard solution. Five different concentrations of Cd (0.2, 0.5, 2, 4, and 10 μg/L) were prepared. The square of the correlation coefficient (R2) was 0.9996, and linearity was considered satisfactory.
Limit of detection and limit of quantification
Blank samples were used to estimate the limit of detection (LOD) and the limit of quantification. The LOD was calculated as the triple standard deviation (SD) of the signal obtained from 11 independently prepared reagent blanks, and this value was 0.001. The LOQ was calculated as 10× SD, and this value was 0.004.
Quality control and assurance
For analytical quality assurance, blanks and duplicate samples were used during the process. An internal standard was added to the blanks, calibration standards, and samples, respectively, to compensate for the matrix effects and signal drift. The recovery rates of the internal standard solutions ranged from 92.6% to 103.7%. To measure the accuracy of the method, the mean recovery rate of the golden thread was determined. A known concentration of Cd standard solution was added to 0.5 g of the samples (n = 6), and the mean recovery ratio was 102.8%.
Total contents, bioaccessible contents, and the bioaccessibility of cadmium in golden thread
The maximum permissible limit according to the Chinese Pharmacopeia of Cd for herbal medicines is 0.3 mg/kg. However, the total concentration of Cd in the six batches of samples was determined to range from 3.203 to 5.723 mg/kg [Table 2]. The mean Cd concentration in six batches of the golden thread was approximately 14-fold that of the maximum permissible limit of Cd in Pharmacopoeia of People's Republic of China (PPRC), and this led to the necessity of assessing the bioaccessibility of Cd in golden thread. The bioaccessible level of Cd in golden thread provides a better index than does the total content to represent the actual level of Cd available to organisms. The bioaccessible concentration of Cd in the gastric phase in six batches of samples ranged from 1.142 to 1.931 mg/kg. The bioaccessibility of Cd in the intestinal phase was lower than that in the gastric phase, and this was consistent with previous environment analysis studies., This discovery resulted from complex factors, and the major factors could be the precipitation of the dissolved Cd due to an increase in the pH (from 2.0 to 7.0). Furthermore, after uptake by Caco2 cells, the bioaccessible concentration of Cd in six batches of samples ranged from 0.601 to 0.882 mg/kg. Compared to the bioaccessible content, bioaccessibility can better represent the phenotype of Cd properties and the exposure forms. After uptake by Caco2 cells, the bioaccessibility of Cd in six batches of the golden thread was determined to range from 42.36% to 59.73%.
Several studies have reported that there is a distinctive disparity in the bioaccessibility of heavy metals in various types of foods. For example, Cai et al. revealed that lettuce possessed the highest Fe bioaccessibility (92.2%), while spinach exhibited the lowest Fe bioaccessibility (54.6%). In regard to Zn, leaf lettuce possessed the highest bioaccessibility (33.1%), while the bioaccessibility of Zn in potato and white radish was quite low. For different foods, the differences in the bioaccessibility of a specific metal could be due to the role of different matrices and the speciation of metals in the food. Therefore, examining the bioaccessibility of diverse heavy metals in different types of TCMs would provide an interesting topic for further research.
The uptake of Cd by Caco2 cells is influenced by many factors. First, the gene expression in response to Cd in Caco2 cells is directly associated with uptake efficiency. For example, the augmented transport of Cd was attributed to an up-regulation of MRP1 gene expression. In addition, uptake of Cd is related to the activity of its ionic form in solution. Studies have shown that unstable chemical forms of Cd (such as Cd chloride complex) could be more easily taken up by cells. In addition, pH, temperature, and other environmental factors markedly influenced the bioaccessibility of Cd. Moreover, the uptake of Cd could be correlated to the levels of essential metals. As Cd is a nonessential metal, a specific carrier protein for transporting Cd is unlikely to exist. However, Cd can be transported into cells by proteins/channels specific for other essential metals (calcium, iron, and zinc). When these nutrient metals are in short supply, the absorption and toxicity of Cd are enhanced.
Health risk assessment
To evaluate the potential health risks, the THQ of Cd in golden thread was calculated based on total concentration and bioaccessible concentration, and these were expressed as THQb and THQt, respectively [Figure 1]. The results revealed that the THQt values of Cd in six batches of golden thread ranged from 1.05 to 1.88. However, the THQb values of the golden thread samples were significantly lower than those calculated based on the total levels, and these values ranged from 0.59 to 0.85 for six batches of samples. The maximum theoretical value of Cd content in golden thread was 5.8 mg/kg when the THQ value was 1 based on the results of bioaccessibility in this study. Bioaccessibility is of high significance, as it offers a more precise assessment of the health risks of heavy metals to humans and may help to avoid the overestimation of health risks from heavy metals and thus unnecessary government intervention and the waste of TCM resources. To the best of our knowledge, we are the first to perform health risk assessment using an in vitro PBET digestion/Caco-2 cell system and bioaccessible Cd in golden thread. The use of this system enables us to obtain more accurate and objective results. After the adjustment for bioaccessibility, the THQb values of Cd in all six batches of analyzed samples were <1, indicating a lower and acceptable health risk. Taken together, our study results suggest that health risks should be evaluated based on the bioaccessible Cd content rather than on the total levels of Cd in the environment. Bioaccessibility measurements of heavy metals in TCMs are critically important to scientifically assess their risk in the real world.
| Conclusion|| |
In this study, we developed a method to measure the bioaccessibility of Cd in golden thread based on a system utilizing in vitro PBET digestion/Caco2 cells, and we further established a novel health risk assessment strategy for Cd in TCM based on bioaccessibility. Our key findings revealed that bioaccessibility is of high significance, as it offers a more accurate and scientific health risk assessment of contaminants to humans. All the THQ values of Cd in the six batches of analyzed samples were <1 after adjustment based on bioaccessibility. Health risks should be evaluated on the basis of the bioaccessible Cd content rather than on the total Cd levels, and importantly, an overestimation of risks or waste of TCM resources may be avoided. The refined and realistic health risk assessment strategy based on bioaccessibility achieved using in vitro PBET digestion/Caco2 cells has provided further confidence regarding the human consumption of golden thread.
This work was financially supported by the 13th Five-Year National Significant New Drugs Creation Feature Subjects grant (2018ZX09735006) and by the Project for Medicine and Medical Instruments Review and Approval System Reform grant (ZG2016-1).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]