Little data are available about the extent of bio-accumulation of heavy metals in traditional animal medicines (TAMs). In the present study, the oral bioavailability of cadmium (Cd) in earthworms and leeches was investigated through in vitro PBET digestion/Caco2 and MDKC cell models. We are the first to create an innovative assessment strategy which has capacity to offer a more precise evaluation of Cd-associated health risks in TAMs, by combinational usage of bioavailable Cd levels, the duration and frequency of the exposure to TAMs obtained by questionnaire data, as well as safety factor of TAMs. Our data showed that the percentage of bioavailability for Caco-2 cells in earthworms and leeches ranged from 3.29–14.17% and 4.32–12.61%, respectively. The percentage of bioavailability of MDCK cells in earthworms and leeches ranged from 4.83–15.74% and 6.53–15.04%, respectively. After adjusting by the bioavailability of Cd to target hazard quotient (THQ), excitingly, our findings manifested that the health risks induced by the ingestion of earthworms and leeches were acceptable in the clinic. Our novel strategy has capacity to offer a more realistic and accurate assessment of Cd-associated health risks in TAMs. Besides, our key findings suggest that bioavailability characterization cannot be ruled out and health risks should be assessed on the basis of the bioavailable Cd levels rather than total levels.
Research Article
Novel risk assessment of Cd in earthworms and leeches based on bioavailability through in vitro digestion/Caco-2 and MDCK cells
https://doi.org/10.21203/rs.3.rs-340887/v1
This work is licensed under a CC BY 4.0 License
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Bioavailability
Traditional animal medicines
Risk assessment
In vitro digestion/Caco-2 cell model
In vitro digestion/MDCK cell model
Cd
- Oral bioavailability of Cd in traditional animal medicines (TAMs) was investigated.
- In vitro PBET digestion/Caco2 and MDKC cell models were developed suited to the matrix of TAMs.
- A novel health risk assessment model for TAMs was created through bioavailability characterization.
Traditional animal medicine (TAM) is a significant type of traditional Chinese medicine (TCM). Given the unique priority of curative discoveries, TAMs have been extensively used for treating a variety of diseases in Chinese clinics for a long history. Both earthworms and leeches are commonly used TAMs, as thoroughly described in ancient literature 1700 year ago (Takahashi et al., 2010). In addition, there has been a long tradition of using earthworms and leeches for clinical purposes since ancient Egypt and medieval Europe (Whitaker et al., 2004). The proposed therapeutic benefits of earthworms include anti-arteriosclerosis, anti-tumor, anti-thrombotic, anti-arrhythmic, immune-modulatory anti-hypertension, and diuresis (Huang et al., 2018). The biological activities of leeches include anti-hypertension, relieving amenorrhea, relieving injuries, cardiovascular protective activities, and anti-cancer effects (Pan et al., 2006).
Cadmium (Cd), a ubiquitous environmental pollutant, is a global concern because of its detrimental effects on the environment and health risks to human bodies (Khana et al., 2017; Tang et al., 2017). Chronic exposure to excessive Cd can induce a series of adverse effects on humans, including obstructive pulmonary disease, reproductive deficiencies, kidney dysfunction, diabetes, osteoporosis, and fractures (Ngo et al., 2012; Chen et al., 2014; Zhang et al. 2014). Previous studies have reported that the content of Cd in TAMs was of great concern (Zuo et al., 2019). The ingestion of TAMs contaminated by Cd through food chains causes the accumulation of this toxin in human organisms, posing potential health risks to humans.
Generally, most studies on the risks associated with Cd have focused on the total Cd contents, providing valuable data on the overall contamination degree of this toxic element (Li et al. 2014; Nemati et al. 2014; Ghaleno et al. 2015; Cheng et al. 2017; Zuo et al. 2020a; Zuo et al. 2020b). However, these measurements did not reflect the actual Cd exposure levels to the human body, leading to higher exposure levels and an unreasonable overestimation of the risks to public health. Thus, it is essential to better understand the concept of bioavailability of Cd to assess its risks to humans, which involves investigating the oral bioaccessibility and bioavailability of Cd in TAMs.
Bioaccessibility is defined as the portion of pollutants that can be released from the matrix to the intestinal tract and has the potential to be absorbed by the human body during digestion (Liu et al. 2018). Recently, a variety of in vitro digestion approaches have been created to examine the bioaccessibility of deleterious elements in soil and food, including the model of PBET, SBET, UBM, etc (Denys et al., 2012; Wragg et al., 2011; Ruby et al., 1996). Compared with in vivo methods, which are time consuming, expensive, and subject to ethical complications, these in vitro digestion models have considerable advantages, including simplicity, high effectiveness, low cost, energy savings, and easy reproducibility (Koch et al., 2007).
Moreover, it should be mentioned that bioavailability is also quite significant. Bioavailability embodies the fraction actually absorbed by organisms and directly used for physiological functions; thus, comparatively, it can provide more reliable and accurate oral exposure (Andre et al., 2015). The Caco-2 cell line, derived from colon carcinoma, has been widely accepted as an in vitro model for mimicking the absorption process of the human intestinal epithelium barrier in studying the transport and retention behaviors of a variety of substances (Balimane et al., 2000). After differentiation in culture, Caco-2 cells represent the biochemical and morphological characteristics of small intestine cells, involving tight junctions, polarity, specific enzymes, and transport systems (Rousset, 1986). However, the tight junctions of this type of cell are actually tighter than those in enterocytes, thus lessening the permeability of compounds with substantial paracellular absorption (Saitoh et al., 2004; Takenaka et al., 2014). Another type of cell lines, MDCK cells, which are originally from the collecting duct of the canine kidney, are typical secretory epithelial cell lines with non-tumor features (Husted et al., 1986). Apart from being used for research on the morphology and function of renal tubular epithelial cells, MDCK cells are widely recognized as an in vitro tool for the evaluating drug absorption and metabolism by the Food and Drug Administration, European Medicines Agency, and other authorities (Volpe, 2008). To the best of our knowledge, scarce data is available on the bioavailability of Cd in TAMs. Additionally, little information is present related to the health risks of bioavailable Cd in TAMs. Therefore, it is of great significance to establish bioavailability models of TAMs to improve risk assessment of contaminants to uniform their clinical usage.
Considering the unique characterizations of TAMs, increasingly popular usage of TAMs worldwide, as well as associated public health concerns, our effort aimed at (1) investigating the contents of total Cd in earthworms and leeches using inductively coupled plasma-mass spectroscopy (ICP-MS), (2) unveiling the bioaccessibility of Cd in earthworms and leeches after simulating human in vitro digestion using a PBET model, (3) analyzing the cellular uptake of Cd in bioavailable fractions using the Caco-2 and MDCK cell models, and (4) creating the first assessment strategy suited to TAMs based on exposure to bioavailable Cd, by which we sought to provide innovative insights into health risks and safety usage of TAMs.
Samples and reagents
Thirteen batches of earthworms and nine batches of leeches were collected from TCM markets and retail pharmacies from 2018 to 2019 in the provinces of Anhui, Guangxi, Hainan, Zhejiang, Jiangsu, Yunnan, Heilongjiang, Shandong, and Shanghai City (Table 1). All samples of earthworms and leeches were authenticated by Dr. Kun-zi Yu.
|
Type |
No. |
Batch No. |
Location |
Source |
|---|---|---|---|---|
|
Earthworm |
1 |
DL-1 |
Anhui |
Pharmacy |
|
2 |
DL-2 |
Anhui |
TCM market |
|
|
3 |
DL-3 |
Shanghai |
Pharmacy |
|
|
4 |
DL-4 |
Shanghai |
Pharmacy |
|
|
5 |
DL-5 |
Shanghai |
TCM market |
|
|
6 |
DL-6 |
Shanghai |
TCM market |
|
|
7 |
DL-7 |
Guangxi |
Pharmacy |
|
|
8 |
DL-8 |
Guangxi |
TCM market |
|
|
9 |
DL-9 |
Guangxi |
TCM market |
|
|
10 |
DL-10 |
Hainan |
Pharmacy |
|
|
11 |
DL-11 |
Hainan |
Pharmacy |
|
|
12 |
DL-12 |
Hainan |
Pharmacy |
|
|
13 |
DL-13 |
Zhejiang |
Pharmacy |
|
|
Leech |
1 |
SZ-1 |
Jiangsu |
Pharmacy |
|
2 |
SZ-2 |
Jiangsu |
TCM market |
|
|
3 |
SZ-2 |
Yunnan |
TCM market |
|
|
4 |
SZ-2 |
Yunnan |
TCM market |
|
|
5 |
SZ-2 |
Shandong |
Pharmacy |
|
|
6 |
SZ-2 |
Shandong |
Pharmacy |
|
|
7 |
SZ-2 |
Shandong |
TCM market |
|
|
8 |
SZ-2 |
Heilongjiang |
Pharmacy |
|
|
9 |
SZ-2 |
Heilongjiang |
TCM market |
Double-deionized water was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). The Cd standard solution was purchased from the National Standard Material Research Center (Beijing, China). The internal standard solution containing In (m/z = 115, 100.0 µg/mL) and tuning solutions containing Li, Y, Ce, Tl, and Co were got from Agilent (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 cell cultures associated with Caco-2 and MDCK cell models, fetal bovine serum (FBS), dulbecco’s modified Eagle’s medium (DMEM) containing glucose, and 0.25% trypsin/EDTA were obtained from Gibco (Carlsbad, CA, USA).
Determination Of The Total Cd Concentrations Via Icp-ms
0.5g homogenous powder of earthworms and leeches was placed into microwave digestion vessels for digestion with 8.0 mL of HNO3. After digestion, excessive acid in the digestion vessels was dispelled. The digestion solution was then diluted to 50.0 mL with Milli-Q water. The Cd contents were analyzed based on our reported methods (Zuo et al., 2019) using the Agilent 7700X ICP-MS. For quality assurance, blanks and duplicates were carried out during the determination process. The internal standard In (m/z 115) was added to the blanks, calibration standards, and samples to compensate for the signal drift and matrix effects. The mean recovery rate of the internal standard solution was 106.9 ± 3.7%. The mean recovery rate of the earthworm (n = 9) was also determined in order to control the accuracy of the method, which was 104.6 ± 45.2 %.
In Vitro Digestion
The bioaccessibility of Cd in the earthworm and leech samples was investigated by applying the in vitro PBET model according to our published methods (Zuo et al.,2020c). The model consisted of two compartments involving a gastric and an intestinal extraction phase (Ruby et al., 1996). Briefly, during the gastric extraction stage, 0.5 g of the samples was mixed with 50 mL simulated gastric solution (pH 2.0 adjusted by HCl). Then, the mixed solutions were incubated at 37°C, shaken for 1 h and centrifuged 5 min to collect the supernatant. Further, 5 mL of nitric acid was mixed with the concentrated supernatant for digestion. Finally, the 50 mL digested solution as the gastric fraction was analyzed by ICP-MS.
In the intestinal extraction stage, the residue from the gastric extraction stage was added to 50 mL simulated intestinal solution containing pancreatin and bile salts (pH 7.0, adjusted by NaHCO3). The solution was incubated and shaken at 37°C for 4 h, centrifuged to collect the supernatant, and concentrated to 3 mL. Then Nitric acid (5 mL) was added to the concentrated supernatant for digestion. Finally, the 50 mL digested solution as the intestinal fraction was measured by ICP-MS.
The bioaccessibility of Cd in both the gastric and intestinal phases was calculated by the following equation:
Caco-2 And Mdck Cell Model
Human colon adenocarcinoma Caco-2 cells and MDCK cells were purchased from Shanghai Institutes for Biological Science of the Chinese Academy of Medical Sciences (Shanghai, China). Both cell types were maintained in DMEM supplemented with 10% (v/v) FBS and 2.3 g/L sodium bicarbonate. These two types of cells were incubated in the condition of 95% air and 5% CO2 at 37°C. The culture medium was refreshed every 2 d, and the cells were transferred when the cell density reached 80% confluence using 0.25% trypsin/EDTA.
Cell differentiation and uptake tests were carried out in a two-chamber well (apical and basolateral) with polyester membrane (12 mm diameter, pore size 0.4 µm; Millipore, Milford, MA, USA). In this system, Caco-2 cells and MDCK cells were seeded at 5.0× 104 and 4×105 cells/mL, respectively. To achieve mature cells and differentiated confluent cell monolayers, the Caco2 cells and MDCK cells were incubated for 21 days and 4 days, respectively, during which the culture medium was refreshed every 2 d. The transepithelial electrical resistance (TEER) measured by Millicell ERS (WPI Corporation, USA) was the used as an indicator of the integrity of the cell monolayer. Only monolayers with TEER > 200 Ω/cm2 were selected for the uptake tests.
The uptake tests were carried out at 21 d and 4 d post seeding for the Caco-2 and MDCK cells, respectively. A 0.5 mL portion of the intestinal fraction was added to the apical side, and 0.5 mL of FBS-free DMEM was added to the basolateral side. After 4 h of incubation at 37°C (5% CO2), the mediums from the apical and basal compartments were harvested separately to measure the transport of Cd across the monolayer. Then, cell monolayers were lysed and microwave digested. The Cd content in the cell lysate was ready for retention analysis using ICP-MS. Control cells were prepared for each assay.
The cellular uptake of Cd was expressed as the content of Cd absorbed by the Caco-2 or MDCK cells (sum of retention and transport) from the intestinal fraction divided by the Cd content in the intestinal fraction. The uptake of Cd was calculated using the following formula:
The bioavailability of Cd was calculated as the content of Cd absorbed by the Caco-2 or MDCK cells (sum of retention and transport) from the intestinal fraction divided by the total Cd content in TAMs. The bioavailability of Cd was calculated as follows:
Health risk assessment
To evaluate the human health risks caused by exposure to Cd stemming from the consumption of TAMs , the value of target hazard quotient (THQ) was calculated using the following formula:
According to our questionnaire data, the exposure frequency (EF), the exposure duration (Ed), and the safety factor (SF) for TAM was 90 days/year, 20 years and 10, respectively. Besides the average body weight (W) and average time exposed to TAMs (AT) was 60kg and 365 days/year× 70 years, respectively (Mahmood et al. 2013). IR is the ingestion rate of TAMs, according to the maximum daily ingestion rate recorded in the Chinese Pharmacopoeia (The Pharmacopoeia Commission of the People's Republic of China, 2015). C is the total Cd contents in earthworms or leeches before extraction (mg/kg). BA is the bioavailability of Cd calculated by equation (3). RfD is the oral reference dose recommended by the US EPA for Cd, which is 1 μg/kg bw/day (Mahmood et al. 2013). If THQ is less than 1, no significant hazardous health effects resulting from exposure to TAMs are expected. If THQ is larger than 1, the health risk of the exposed population cannot be ignored.
Total concentration of Cd in earthworms and leeches
The levels of Cd varied remarkably from earthworms to leeches. This was likely caused by the organisms’ selective bio-accumulation of different TAMs for heavy metals, their specific living environments, and their respective locations in food chains (Vicente-Martorell et al., 2009; Wang et al., 2017; Amoozadeh et al., 2014). Total contents of Cd in different batches of earthworms and leeches were illustrated in Table 2. The mean levels of Cd in 13 batches of earthworms and 9 batches of leeches were 12.649 and 1.506 mg/kg, respectively. Some previous studies have reported Cd contents in the environment and food. Gu indicated that the mean Cd levels in marine organisms at different trophic levels in the South China Sea ranged from 0.04 to 0.53 mg/kg, which were much lower than those in earthworms or leeches (Gu et al., 2018). Cui reported that the average Cd concentration in fingered citron was 0.121 mg/kg, which was 0.01 times that of earthworms and 0.08 times of leeches in the present study (Cui et al., 2018). The average Cd levels in hops (Humulus lupulus L.) from different geographical origins were 0.03 to 0.04 mg/L, which were notably lower than those in earthworms or leeches in the present study (Liu et al., 2019). Thus, it is necessary to study the bioaccessibility of Cd in TAMs owing to their higher concentrations.
|
Types |
No. |
Total |
Gastric phase(G) |
Intestinal phase(I) |
|---|---|---|---|---|
|
Earthworm |
1 |
24.774 ± 1.242 |
10.249 ± 0.287 |
7.377 ± 0.028 |
|
2 |
11.111 ± 0.393 |
4.591 ± 0.083 |
3.444 ± 0.012 |
|
|
3 |
33.606 ± 1.875 |
12.624 ± 0.281 |
9.160 ± 0.093 |
|
|
4 |
13.406 ± 0.986 |
5.688 ± 0.309 |
4.428 ± 0.036 |
|
|
5 |
8.319 ± 0.238 |
4.168 ± 0.298 |
4.141 ± 0.029 |
|
|
6 |
16.691 ± 1.267 |
6.581 ± 0.398 |
6.309 ± 0.328 |
|
|
7 |
5.216 ± 0.087 |
2.376 ± 0.082 |
2.051 ± 0.028 |
|
|
8 |
8.215 ± 0.036 |
4.863 ± 0.093 |
2.607 ± 0.093 |
|
|
9 |
11.184 ± 0.023 |
5.597 ± 0.092 |
3.168 ± 0.183 |
|
|
10 |
4.625 ± 0.292 |
2.473 ± 0.329 |
1.724 ± 0.043 |
|
|
11 |
6.724 ± 0.983 |
2.030 ± 0.498 |
2.017 ± 0.072 |
|
|
12 |
9.682 ± 0.129 |
5.679 ± 0.086 |
3.182 ± 0.183 |
|
|
13 |
10.885 ± 1.875 |
6.893 ± 0.287 |
3.370 ± 0.239 |
|
|
Leech |
1 |
1.896 ± 0.065 |
0.876 ± 0.459 |
0.704 ± 0.029 |
|
2 |
1.692 ± 0.082 |
0.896 ± 0.028 |
0.608 ± 0.098 |
|
|
3 |
1.329 ± 0.069 |
0.675 ± 0.011 |
0.447 ± 0.023 |
|
|
4 |
1.682 ± 0.025 |
0.876 ± 0.223 |
0.513 ± 0.038 |
|
|
5 |
1.876 ± 0.093 |
0.674 ± 0.039 |
0.446 ± 0.026 |
|
|
6 |
1.523 ± 0.086 |
0.765 ± 0.092 |
0.544 ± 0.048 |
|
|
7 |
0.986 ± 0.022 |
0.587 ± 0.014 |
0.345 ± 0.018 |
|
|
8 |
0.893 ± 0.013 |
0.398 ± 0.002 |
0.230 ± 0.002 |
|
|
9 |
1.679 ± 0.094 |
0.896 ± 0.024 |
0.643 ± 0.038 |
Bioaccessible concentration and bioaccessibility of Cd in earthworms and leeches
Table 2 showed bioaccessible Cd in gastric (G) and intestinal (I) fractions. The bioaccessible concentrations of Cd in the gastric phase were 2.030-12.624 mg/kg for earthworms and 0.398–0.896 mg/kg for leeches, respectively. The bioaccessible contents of Cd in the intestinal phase ranged from 1.724 to 9.160 mg/kg for earthworms and from 0.230 to 0.704 mg/kg for leeches, respectively. The bioaccessibility of Cd in the intestinal phase was lower than that in the gastric phase (Fig. 1), which was in accordance with previous studies. For instance, Intawongse and Dean found that the bioaccessibility of Cd in radish roots in the small intestinal phase (24.8%) was lower than that in the gastric phase (54.9%). This discovery may result from complex factors, involving the increased pH value from the gastric phase to the intestinal phase as well as the additional precipitation of Cd owing to the supplementation of bile salts and secretin in the intestinal phase (Ruby et al. 1993). In the present study, the bioaccessibility of Cd in the gastric phase was 30.19%-66.33% for earthworms and 35.93%-59.53% for leeches, respectively. The bioaccessibility of Cd in the intestinal phase was 27.26%-49.78% for earthworms and 23.80-38.27% for leeches, respectively. Some published data on the bioaccessibility of Cd associated with the environment are currently available (Karadas and Kara, 2011; Pelfrêne et al., 2013). Generally, the bioaccessibility in the present study was comparable with previous studies. Interestingly, studies have reported that the bioaccessibility of Cd is higher than that of many other toxic metals (Poggio et al., 2009; Pelfrêne et al., 2013).
Retention and transport of Cd in Caco-2 and MDCK cells
Bioaccessibility is only associated with the maximum oral bioavailability, because absorption in the intestinal epithelia cells could decrease the portion of Cd that reaches the body circulation (Versantvoort et al., 2005). Thus, in the present study, the in vitro PBET digestion was advanced by coupling with Caco-2 or MDCK cell cultures to offer a more accurate estimate of the uptake (retention + transport) in the intestine. Moreover, a mass balance was tested by comparing the levels of Cd originally added to the cells with the contents of Cd obtained after the cell uptake assays. In the case of Caco2 cells, the mass balance showed that the recovery rates were from 70.53–100.79%. In the case of MDCK cells, the recoveries ranged between 70.40% and 95.47%. In the cell monolayer of Caco-2 cells, the percentage of retention for Cd ranged from 3.69–24.10% in earthworms and from 5.15–16.05% in leeches (Table 3). In the MDCK cell monolayer, the retention percentage of Cd varied from 7.26–22.63% in earthworms and from 15.01–19.46% in leeches (Table 4). For transport in the Caco-2 cells, the ratios of transport for Cd were from 4.26–20.81% in earthworms and from 3.19–19.40% in leeches (Table 3). In MDCK cells, the transport ratios ranged from 7.81–25.63% in earthworms and from 11.94–24.07% in leeches (Table 4).
|
Sample |
No. |
Apical compartment |
Cell monolayer (retention) |
Basolateral compartment (transport) |
Total cellular uptake (%) |
Bioavailability(%) |
|||
|---|---|---|---|---|---|---|---|---|---|
|
mg/kg |
% |
mg/kg |
% |
mg/kg |
% |
||||
|
Earthworm |
1 |
3.679 |
49.87 |
0.876 |
11.87 |
0.763 |
10.34 |
22.22 |
6.62 |
|
2 |
2.786 |
80.88 |
0.127 |
3.69 |
0.238 |
6.91 |
10.60 |
3.29 |
|
|
3 |
6.813 |
74.38 |
1.539 |
16.80 |
0.881 |
9.61 |
26.42 |
7.20 |
|
|
4 |
2.378 |
53.70 |
1.067 |
24.10 |
0.189 |
4.26 |
28.36 |
9.37 |
|
|
5 |
1.942 |
46.90 |
0.896 |
21.64 |
0.283 |
6.83 |
28.47 |
14.17 |
|
|
6 |
3.877 |
61.46 |
0.686 |
10.87 |
0.485 |
7.69 |
18.57 |
7.02 |
|
|
7 |
1.048 |
51.10 |
0.137 |
6.68 |
0.427 |
20.81 |
27.49 |
10.81 |
|
|
8 |
1.463 |
56.13 |
0.368 |
14.12 |
0.141 |
5.41 |
19.53 |
6.20 |
|
|
9 |
1.856 |
58.59 |
0.216 |
6.82 |
0.320 |
10.10 |
16.91 |
4.79 |
|
|
10 |
0.875 |
50.77 |
0.236 |
13.69 |
0.174 |
10.10 |
23.79 |
8.86 |
|
|
11 |
1.179 |
58.45 |
0.206 |
10.21 |
0.198 |
9.82 |
20.04 |
6.01 |
|
|
12 |
1.982 |
62.29 |
0.402 |
12.63 |
0.308 |
9.68 |
22.31 |
7.33 |
|
|
13 |
1.664 |
49.38 |
0.439 |
13.03 |
0.383 |
11.36 |
24.39 |
7.55 |
|
|
Leech |
1 |
0.368 |
52.27 |
0.113 |
16.05 |
0.126 |
17.90 |
33.95 |
12.61 |
|
2 |
0.391 |
64.36 |
0.068 |
11.19 |
0.059 |
9.77 |
20.96 |
7.53 |
|
|
3 |
0.233 |
52.08 |
0.035 |
7.82 |
0.052 |
11.60 |
19.43 |
6.54 |
|
|
4 |
0.348 |
67.89 |
0.058 |
11.30 |
0.094 |
18.32 |
29.62 |
9.04 |
|
|
5 |
0.330 |
73.92 |
0.023 |
5.15 |
0.058 |
12.99 |
18.14 |
4.32 |
|
|
6 |
0.282 |
51.84 |
0.082 |
15.06 |
0.029 |
5.33 |
20.39 |
7.29 |
|
|
7 |
0.192 |
55.66 |
0.052 |
15.08 |
0.011 |
3.19 |
18.26 |
6.39 |
|
|
8 |
0.128 |
55.73 |
0.023 |
10.01 |
0.045 |
19.40 |
29.41 |
7.56 |
|
|
9 |
0.386 |
60.07 |
0.068 |
10.58 |
0.065 |
10.12 |
20.70 |
7.92 |
|
|
Sample |
No. |
Apical compartment |
Cell monolayer (retention) |
Basolateral compartment (transport) |
Total cellular uptake (%) |
Bioavailability(%) |
|||
|---|---|---|---|---|---|---|---|---|---|
|
mg/kg |
% |
mg/kg |
% |
mg/kg |
% |
||||
|
Earthworm |
1 |
3.746 |
50.78 |
1.237 |
16.77 |
1.143 |
15.49 |
32.26 |
9.61 |
|
2 |
1.756 |
50.98 |
0.523 |
15.18 |
0.729 |
21.16 |
36.34 |
11.27 |
|
|
3 |
3.687 |
40.25 |
1.364 |
14.89 |
2.347 |
25.63 |
40.52 |
11.04 |
|
|
4 |
2.118 |
47.83 |
1.002 |
22.63 |
1.108 |
25.01 |
47.64 |
15.74 |
|
|
5 |
1.658 |
40.04 |
0.527 |
12.73 |
0.730 |
17.63 |
30.36 |
15.11 |
|
|
6 |
3.298 |
52.28 |
0.458 |
7.26 |
0.981 |
15.55 |
22.81 |
8.62 |
|
|
7 |
1.251 |
61.00 |
0.259 |
12.63 |
0.269 |
13.12 |
25.74 |
10.12 |
|
|
8 |
1.189 |
45.61 |
0.267 |
10.24 |
0.388 |
14.90 |
25.14 |
7.98 |
|
|
9 |
1.249 |
39.44 |
0.345 |
10.89 |
0.769 |
24.29 |
35.18 |
9.96 |
|
|
10 |
0.988 |
57.34 |
0.387 |
22.45 |
0.242 |
14.04 |
36.49 |
13.60 |
|
|
11 |
1.148 |
56.91 |
0.167 |
8.28 |
0.158 |
7.81 |
16.09 |
4.83 |
|
|
12 |
1.368 |
42.99 |
0.548 |
17.22 |
0.753 |
23.65 |
40.87 |
13.43 |
|
|
13 |
1.717 |
50.95 |
0.423 |
12.55 |
0.651 |
19.32 |
31.87 |
9.87 |
|
|
Leech |
1 |
0.351 |
49.91 |
0.137 |
19.46 |
0.124 |
17.61 |
37.07 |
13.77 |
|
2 |
0.324 |
53.33 |
0.118 |
19.42 |
0.112 |
18.44 |
37.86 |
13.59 |
|
|
3 |
0.174 |
38.91 |
0.075 |
16.76 |
0.102 |
22.80 |
39.56 |
13.32 |
|
|
4 |
0.157 |
30.53 |
0.098 |
19.10 |
0.112 |
21.83 |
40.92 |
12.49 |
|
|
5 |
0.196 |
43.91 |
0.067 |
15.01 |
0.055 |
12.42 |
27.43 |
6.53 |
|
|
6 |
0.274 |
50.30 |
0.098 |
18.00 |
0.131 |
24.07 |
42.07 |
15.04 |
|
|
7 |
0.203 |
58.85 |
0.057 |
16.53 |
0.041 |
11.94 |
28.47 |
9.96 |
|
|
8 |
0.089 |
38.75 |
0.034 |
14.80 |
0.052 |
22.54 |
37.35 |
9.60 |
|
|
9 |
0.303 |
47.15 |
0.112 |
17.43 |
0.135 |
20.98 |
38.41 |
14.70 |
|
Uptake and bioavailability of Cd in Caco-2 and MDCK cells
The fraction of the total cellular uptake (retention + transport) for Caco-2 and MDCK cells in earthworms ranged from 10.60–28.47% and 16.09–47.64%, respectively. In leeches, the total cellular uptake fractions ranged from 18.14–33.95% and from 27.43 % to 42.07%, respectively. Our study compared the cellular uptake of Cd in earthworms and leeches between Caco-2 and MDCK cell lines, and the results revealed that in both types of TAMs, the uptake of Cd in MDCK cells was higher than that in Caco-2 cells cells; this was probably caused by the difference in looseness of the tight junctions (Lee et al., 2018). Moreover, the percentage of bioavailability for Caco-2 cells in earthworms and leeches was from 3.29–14.17% and 4.32 % to 12.61%, respectively. The percentage of bioavailability of MDCK cells in earthworms and leeches ranged from 4.83 % to 15.74% and 6.53 % to 15.04 %, respectively.
To the best of our knowledge, little valuable data on the bioavailability of detrimental metals in TCMs or TAMs are available, whereas the bioavailability of toxic metal elements associated with food, water, or soil has been investigated in several researches. Generally, the bioavailability of Cd in our study was comparable with those found in previous studies. For example, Fu and Cui reported that the bioavailability of Cd ranged from 4.0–15.9% for the vegetables (Fu and Cui, 2013). However, the bioavailability of Cd in the present study was higher or lower than some other studies reported on CdCl2 solution and infant food (Eklund et al., 2003; Chan et al., 2007). Besides, in an in vivo experiment, the bioavailability of Cd was 0.8% and 0.6% in the kidneys and livers of rats, respectively (Yannai and Sachs, 1993). The uptake of Cd by Caco2 or MDCK cells is influenced by many factors. First, the gene expression of Cd in cells is directly associated with its uptake efficiency. The augmented uptake of Cd was attributed to the upregulation of MRP1 gene expression (Öhrvik et al., 2013). In addition, the uptake of Cd is related to the activity of its ionic form in solution. Studies have demonstrated that labile complexes of Cd enhanced bioavailability of metals in cells, possibly by the mechanism of alleviating diffusive limitations (Verheyen et al., 2012). Moreover, the temperature, pH, and other environmental factors greatly influence the uptake of Cd (Reeves and Chaney, 2008). Additionally, the uptake of Cd can be evidently modulated by the organism’s requirement for essential metals, including Zn, Fe, and Ca. Because Cd is a non-essential element, specific carrier proteins/channels for transporting Cd are unlikely to exist. However, experimental evidence have suggested that proteins of essential metals, such as divalent metal transporter 1, were responsible for the transport of Cd. When these nutrient elements were in short supply, the accumulation and toxicity of Cd are enhanced (Vesey, 2010). Another study by Reeves and Chaney revealed that absorption of Cd from food could be decreased if the calcium contents were high (Reeves and Chaney, 2008). Furthermore, different types of matrixes seemed to play a significant role in the differences of bioavailability (Fu and Cui, 2013).
Excitingly, our study built up an innovative assessment strategy of Cd-associated health risks of TAMs based on in vitro PBET digestion/Caco2 and MDKC cell models for the first time. Moreover, the duration and frequency of exposure to TAMs was acquired from questionnaire information, and the safety factor of TAMs was applied to establish assessment models applicable to TAMs. Taken together, by the comprehensive usage all of the above, we figured out to innovate assessment approaches that were able to scientifically and precisely determine the health risks of Cd in TAMs. Our key findings suggested that health risks should be assessed based on the bioavailable Cd levels rather than the total levels to offer a more realistic and accurate assessment of toxins to humans. Importantly, an overestimation of health risks or wastage of medicine resources may be avoided. We hope the novel health risk assessment strategy applicable to TAMs based on in vitro digestion/cellular uptake opened a window for the bio-accumulation of Cd in organisms as well as toxicological evaluation of TAMs, in order to improve public health by guiding the usage of TCMs in the clinic.
TAM: Traditional animal medicines; PBET: the physiologically based extraction test; ICP-MS: inductively coupled plasma mass spectrometry; FBS: fetal bovine serum; DMEM: Dulbecco’s modified eagle medium; TEER: transepithelial electrical resistance; THQ: target hazard quotient
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
All data are fully available without restriction.
Declaration of competing interests
The authors declare that they have no competing interests.
Acknowledgement
This work was financially supported by the 13th 5 Year National Significant New Drugs Creation Feature Subjects (2018ZX09735006) and Youth Fund by National Institutes for Food and Drug Control (2020A3).
Authors' contributions
SCM and LCH designed the study. TTZ, FYL, and FG conducted the experiments. TTZ analyzed the data. TTZ wrote the manuscript. SCM, LS, SXX and BL revised the manuscript. All authors read and approved the final manuscript.
Amoozadeh, E., Malek, M., Rashidinejad, R., Nabavi, S., Karbassi, M., Ghayoumi, R., Ghorbanzadeh-Zafarani, G., Salehi, H., Sures, B., 2014. Marine organisms as heavy metal bioindicators in the Persian Gulf and the Gulf of Oman. Environ. Sci. Pollut. Res. 21, 2386-2395.
Andre, C. M., Evers, D., Ziebel, J., Guignard,C., Hausman, J. F., Bonierbale, M., Zum Felde , T., Burgos, G., 2015. In Vitro Bioaccessibility and Bioavailability of Iron from Potatoes with Varying Vitamin C, Carotenoid, and Phenolic Concentrations. J. Agric. Food. Chem. 63, 9012-9021.
Balimane, P.V., Chong, S., Morrison, R.A., 2000. Current methodologies used for evaluation of instetinal permeability and absorption. J. Pharmacol. Toxicol. Methods. 44, 301-312.
Chan, D.Y., Black, W.D., Hale, B.A., 2007. Cadmium bioavailability and bioaccessibility as determined by in vitro digestion, dialysis and intestinal epithelial monolayers, and compared to in vivo data. J. Environ. Sci. Health A (Part A) 42, 1283-1291.
Chen, X., Wang, K., Wang, Z., Gan, C., He, P., Liang, Y., Jin, T., Zhu, G., 2014. Effects of lead and cadmium co-exposure on bone mineral density in a Chinese population. Bone.63, 76-80.
Cheng, J.L., Zhang, X.H., Tang, Z.W.,Yang, Y.F., Nie, Z.Q., Huang, Q.F.,2017. Concentrations and human health implications of heavy metals in market foods from a Chinese coal-mining city. Environ. Toxicol. Pharmacol. 50, 37-44.
Cui, G.L., Li, L.Y., Tan J., Zhang Y., Niu J., 2018. Assay and Safety Evaluation of Heavy Metals in Citrus medica var. sarcodactylis from Different Areas of Sichuan Province. Chin. J. Trop. Crop. 39(12), 2491-2498.
Denys, S., Caboche, J., Tack, K., Rychen, G., Wragg, J., Cave,M., Jondreville, C., Feidt, C., 2012.Vivo validation of the unified BARGE method to assess the bioaccessibility of arsenic, antimony, cadmium, and lead in soils. Environ. Sci. Technol. 46, 6252-6260.
Eklund, G., Lindén, A., Tallkvist, J., Oskarsson, A., 2003. Bioavailability of cadmium from in vitro digested infant food studied in Caco-2 cells. J. Agric. Food Chem. 51, 4168-4174.
Fu, J., and Cui, Y., 2013. In vitro digestion/Caco-2 cell model to estimate cadmium and lead bioaccessibility/bioavailability in two vegetables: the influence of cooking and additives. Food. Chem. Toxicol. 59, 215-221.
Ghaleno, O. R., Sayadi, M., Rezaei, M., 2015. Potential ecological risk assessment of heavy metals in sediments of water reservoir case study: Chah Nimeh of Sistan. Proc. Int. Acad. Ecol. Environ. Sci. 5(4), 89-96.
Gu, Y.G., Ning, J.J, Ke, C.L., Huang, H.H., 2018. Bioaccessibility and human health implications of heavy metals in different trophic level marine organisms: a case study of the south china sea. Ecotoxicol. Environ. Saf. 163, 551-557.
Huang, J.W., Gao, H.W., Duan, J.F., 2018. Research on chemical composition and pharmacological effects of geosaurus. Guid. J. Tradit. Chin. Med. Pharm. 24, 104-107.
Husted, R.F., Welsh, M.J., Stokes, J.B., 1986. Variability of functional characteristics of MDCK cells. Am. J. Physiol. 250(2 Pt 1), C214-221.
Intawongse, M., Dean, J.R., 2006. Uptake of heavy metals by vegetable plants grown on contaminated soil and their bioavailability in the human gastrointestinal tract. Food. Addit. Contam. 23, 36–48.
Karadas, C., Kara, D., 2011. In vitro gastro-intestinal method for the assessment of heavy metal bioavailability in contaminated soils. Environ. Sci. Pollut. Res. 18,620–628.
Khana, M.A., Khan, S., Khan, A., Alamc, M., 2017. Soil contamination with cadmium, consequences and remediation using organic amendments. Sci. Total. Environ. 601–602, 1591-1605.
Koch, I., McPherson, K., Smith, P., Easton, L., Doe, K.G., Reimer, K.J., 2007. Arsenic bioaccessibility and speciation in clams and seaweed from a contaminated marine environment. Mar. Pollut. Bull. 54,586-594.
Lee, S.G., Kim, J., Park, H., Holzapfel, W., Lee, K.W., 2018. Assessment of the effect of cooking on speciation and bioaccessibility/cellular uptake of arsenic in rice, using in vitro digestion and Caco-2 and PSI cells as model. Food. Chem. Toxicol. 111, 597-604.
Li, Y.Y., Wang, H.B., Wang, H.J., Yin, F., Yang, X.Y.,Hu Y.J.,2014. Heavy metal pollution in vegetables grown in the vicinity of a multimetal mining area in Gejiu, China: total concentrations, speciation analysis, and health risk. Environ. Sci. Pollut. Res.21(21), 12569-12582.
Liu, S.Y., Tian, S.H., Li, K., Wang, L.Q., Liang, T., 2018. Heavy metal bioaccessibility and health risks in the contaminated soil of an abandoned, small-scale lead and zinc mine. Environ. Sci. Pollut. Res. 25(15), 15044-15056.
Liu, Z.C., Wang, Y., Liu, Y.C., 2019. Geographical origins and varieties identification of hops (Humulus lupulus L.) by multi-metal elements fingerprinting and the relationships with functional ingredients. Food. Chem. 289, 522-530.
Mahmood, A., and Malik, R.N., 2013. Human health risk assessment of heavy metals via consumption of contaminated vegetables collected from different irrigation sources in Lahore, Pakistan. Arab. J. Chem. 7(1), 91–99.
Nemati, S., Mosaferi, M., Ostadrahimi, A. & Mohammadi, A., 2014. Arsenic intake through consumed rice in Iran: markets role or government responsibility. Environ. Health. Perspect. 4 (2), 180.
Ngo, D.M., Hough, R.L., Le, T.T., Nyberg, Y., Le, B.M., Nguyen, C.V., Nguyen, M.K., Oborn, I., 2012. Assessing dietary exposure to cadmium in a metal recycling community in Vietnam: age and gender aspects. Sci. Total. Environ. 416, 164-171.
Öhrvik, H., Tydén, E., Artursson, P., Oskarsson, A., Tallkvist, J., 2013. Cadmium transport in a model of neonatal intestinal cells correlates to MRP1 and not DMT1 or FPN1. ISRN. Toxicol. 2013, 1-9.
Pan, H., Gang, H.L., Su, Y.M., 2006. Active components and pharmacological action of leech. Inform. Tradit. Chin. Med. 1, 20-22.
Pelfrêne A., Waterlot, C., Mazzuca, M., Nisse, C., Cuny, D., Richard, A., Denys, S., Heyman, C., Roussel, H., Bidar, G., Douay, F., 2012. Bioaccessibility of trace elements as affected by soil parameters in smelter-contaminated agricultural soils: a statistical modeling approach. Environ. Pollut. 160,130–138.
Poggio, L., Vrščaj, B., Schulin, R., Hepperle, E., Ajmone Marsan, F., 2009. Metals pollution and human bioaccessibility of topsoils in Grugliasco (Italy). Environ. Pollut. 157(2), 680–689.
Reeves, P.G., Chaney, R.L., 2008. Bioavailability as an issue in risk assessment and
management of food cadmium: a review. Sci. Total Environ. 398, 13–19.
Rousseau, M.C., Straif, K., Siemiatycki, J., 2005. IARC carcinogen update. Environ.
Health Perspect. 113, A580.
Ruby,M.V., Davis, A., Schoof, R., Eberle, S., Sellstone, C.M., 1996. Estimation of lead and arsenic bioavailability using a physiologically based extraction test. Environ. Sci. Technol. 30, 422-430.
Saitoh, R., Sugano, K., Takata, N., Tachibana, T., Higashida, A., Nabuchi, Y., Aso, Y., 2004. Correction of permeability with pore radius of tight junctions in Caco-2 monolayers improves the prediction of the dose fraction of hydrophilic drugs absorbed by humans. Pharm. Res. 21, 749-755.
Takahashi, K., Azuma, Y., Shimada, K., Saito, T., Kawase, M., Schaffer, S.W., 2010. Quality and safety issues related to traditional animal medicine: role of taurine. J. BioMed Sci. 17 (Suppl 1), 1–9.
Takenaka, T., Harada, N., Kuze, J., Chiba, M., Iwao, T., Matsunaga, T., 2014. Human
small intestinal epithelial cells differentiated from adult intestinal stem cells as a novel system for predicting oral drug absorption in humans. Drug. Metab.Dispos. 42, 1947-1954.
Tang, W.Z., Zhang, W.Q., Zhao, Y., Zhang, H., Shan, B.Q., 2017. Basin-scale comprehensive assessment of cadmium pollution, risk, and toxicity in riverine sediments of the Haihe Basin in north China. Ecol. Indic. 81, 295-301.
The Pharmacopoeia Commission of the People's Republic of China (2015 Edition). (2015). Beijing, China: Pharmacopoeia of the People's Republic of China, Chinese Medical Science and Technology Publishers.
Verheyen, L., Degryse, F., Niewold, T., Smoldersa, E., 2012. Labile complexes facilitate cadmium uptake by Caco-2 cells. Sci. Total. Environ. 426, 90-99.
Versantvoort, C.H., Oomen, A.G., Van de Kamp, E., Rompelberg, C.J., Sips, A.J., 2005. Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food. Food. Chem. Toxicol. 43, 31–40.
Vesey, D.A., 2010. Transport pathways for cadmium in the intestine and kidney proximal tubule: focus on the inter-action with essential metals. Toxicol. Lett. 198(1), 13-19.
Vicente-Martorell, J.J., Galindo-Riaño, M.D., García-Vargas, M., Granado-Castro, M.D., 2009. Bioavailability of heavy metals monitoring water, sediments and fish species from a polluted estuary. J. Hazard. Mater. 162, 823–836.
Volpe, D.A., 2008. Variability in Caco-2 and MDCK cell-based intestinal permeability assays. J. Pharm Sci. 97(2), 712-725.
Wang, Y.R., Wang, R.M., Fan, L.Y., Chen, T.T., Bai, Y.H., Yu, Q.R., Ying, L., 2017 Assessment of multiple exposure to chemical elements and health risks among residents near Huodehong lead-zinc mining area in Yunnan, Southwest China. Chemosphere. 174, 613–627.
Whitaker, I.S., Rao, J., Izadi, D., 2004. Butler PE: Historical article: Hirudo medicinalis: ancient origins of, and trends in the use of medicinal leeches throughout history. Br. J. Oral. Maxillofac. Surg. 42, 133–137.
Wragg J., Cave, M., Basta, N., Brandon, E., Casteel, S., Denys, S., Gron, C., Oomen, A., Reimer, K., Tack, K., van de Wiele, T., 2011. An inter-laboratory trial of the unified BARGE bioaccessibility method forarsenic, cadmium and lead in soil. Sci. Total. Environ. 409, 4016-4030.
Zhang, W.L., Du, Y., Zhai, M.M., Shang, Q., 2014. Cadmium exposure and its health effects: a 19-year follow-up study of a polluted area in China. Sci. Total. Environ. 470, 224-228.
Zuo, T.T., Li,Y.L., He, H.Z., Jin, H.Y., Zhang, L., Sun, L., Gao, F., Wang, Q., Shen, Y.J., Ma, S.C., He, L.C., 2019. Refined assessment of heavy metal-associated health risk due to the consumption of traditional animal medicines in human. Environ. Monit. Assess. 191, 171-182.
Zuo, T.T., Jin, H.Y., Zhang, L., Liu, Y.L., Nie, J., Chen, B.L., Fang, C.F., Xue, J., Bi, X.Y., Zhou, L., Shen M.R., Shi, S.M., Ma, S.C., 2020a. Innovative health risk assessment of heavy metals in Chinese herbal medicines based on extensive data. Pharmacol. Res.159, 104987-104998.
Zuo, T.T., Qu, H.R., Jin, H.Y., Zhang, L., Luo, F.Y., Yu, K.Z., Gao, F., Wang, Q.,Sun, L., He, H.Z., Ma, S.C., 2020b. Innovative health risk assessments of heavy metals based on bioaccessibility due to the consumption of traditional animal medicines. Environ. Sci. Pollut. Res. 27(18), 22593-22603.