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The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer
Environmental Research 154 (2017) 93–100
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The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer
MARK
⁎
Yingxin Yu , Mengmeng Wang, Kaiqiong Zhang, Dan Yang, Yufang Zhong, Jing An, Bingli Lei, Xinyu Zhang Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China
A R T I C L E I N F O
A BS T RAC T
Keywords: Caco-2 cell monolayer Efflux transporter Influx transporter Polybrominated biphenyl ethers Transepithelial transport
Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH.
1. Introduction Polybrominated diphenyl ethers (PBDEs) are a class of typical persistent organic pollutants (POPs), which can cause adverse effects in the ecosystem and in humans. They were widely used as brominated flame retardants in various consumer products, especially in textiles, plastics, and electronic appliances [10]. As they are not chemically bound to the materials, they can be released from the materials into the environment. Due to their stability and semi-volatility, PBDEs are difficult to degrade in the environment and can be transported long distances [21]. In addition to their high lipophilicity, PBDEs are persistent with the potential for bioaccumulation in organisms and biomagnification through food chains. They have been detected in all types of biota [1,20,29,54]. In the past few years, the levels of PBDEs in biota have increased significantly and PBDEs have been detected in human tissue samples [1,20,41,43]. Toxicity studies have shown that PBDEs result in embryotoxicity, developmental neurotoxicity, and
reproductive toxicity [10,3,7]. The risks due to these chemicals have attracted increasing attention in recent years. Oral ingestion is the main exposure pathway for PBDEs in the non-professional population [27]. Caco-2 cells, derived from human colon carcinoma, express tight junctions, micro villi, and many enzymes and transporters present in the normal human small intestine [40]. These cells can differentiate and polarize, and form a monolayer when cultured under specific conditions. Because of their similar morphology and function to human small intestinal epithelial cells, the Caco-2 cell model is widely used as a tool to investigate the absorption, transport, and metabolism of xenobiotics [15,16,32,44,49]. The efflux transporters, P-glycoprotein (p-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP), which belong to the ATP-binding cassette transporters (ABC transporters), have been shown to pump drugs back to the lumen, and act as a biological barrier, which can affect the bioavailability of
Abbreviations: ABC transporters, ATP-binding cassette transporters; AP, apical; BCRP, breast cancer resistance protein; BL, basolateral; DMSO, dimethyl sulphoxide; EDTA, ethylenediamine tetraacetic acid; GC/MS, gas chromatography/mass spectrometry; MEM, minimum essential medium; MRP, multidrug resistance-associated protein; OATP2B1, organic anion transporting polypeptide B; OATPs, organic anion transporting polypeptides; OCTs, Organic cation transporters; PAHs, polycyclic aromatic hydrocarbons; PBDEs, polybrominated diphenyl ethers; PCBs, polychlorinated biphenyls; P-gp, P-glycoprotein; POPs, persistent organic pollutants; TEER, transepithelial electrical resistance. ⁎ Corresponding author. E-mail address: yuyingxin@staff.shu.edu.cn (Y. Yu). http://dx.doi.org/10.1016/j.envres.2016.12.024 Received 29 August 2016; Received in revised form 2 December 2016; Accepted 20 December 2016 0013-9351/ © 2016 Elsevier Inc. All rights reserved.
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2. Material and methods
was changed to dimethyl sulphoxide (DMSO). Prior to PBDE exposure, the Caco-2 cell monolayer was washed three times with D-Hanks solution. After pre-incubation, the buffer was then gently removed using nitrogen. To investigate the influence of time on transepithelial transport, PBDEs were added into the apical (AP) side or basolateral (BL) side. The volume of medium added to the AP and BL chambers was 0.5 and 1.5 mL, respectively. Following incubation, the solutions from the AP and BL sides were collected and the cells were harvested using 0.25% trypsin and 0.03% EDTA, respectively. PBDEs in the solutions from the AP and BL sides were extracted and determined using gas chromatography/mass spectrometry (GC/MS). For PBDEs in the cells, the harvested cells were disrupted using cell breaking apparatus, and then extracted as described previously. To investigate the influence of PBDE concentrations on transport, four concentrations of PBDE (2.5, 5, 10, and 20 ng/mL) in the medium were used. Various PBDE exposure times (1, 2, 4, 8, 12, and 24 h) at the PBDE concentration of 10 ng/mL were used to study the time effects. In subsequent experiments, the PBDE concentration was set at 10 ng/mL, and the PBDE exposure time was 12 h. The influence of pH on the transport of PBDEs in both directions, i.e., AP to BL and BL to AP, was determined using the following paired pH values in the AP/BL compartments, i.e., 5.5/7.4 and 7.4/7.4. The experiment was carried out at two temperatures (37 and 4 °C) to investigate the influence of energy. To determine the roles of P-gp, MRP, BCRP, and OCTs during transport, 100 μM verapamil [6], 100 μM MK571 [52], 10 μM Ko143 [12], and 50 μM cimetidine [2] were used as inhibitors, respectively. We found that concentrations of DMSO below 0.2% had no significant effect on the viability of Caco-2 cells (data not shown). Therefore the concentrations of DMSO used in the present study were below 0.2%.
2.1. Caco-2 cell culture
2.4. Analytical protocol
Caco-2 cells, purchased from BioHermes Bio & Medical Technology Co., Inc. (Wuxi, China), were cultured in minimum essential medium (MEM) supplemented with 20% fetal bovine serum and 1% penicillin. The cells were grown in a 5% CO2 atmosphere at 37 °C under humidified conditions. The culture medium was replaced every 1–2 days. The cells were passaged using 0.25% trypsin and 0.03% ethylenediamine tetraacetic acid (EDTA) until the cell monolayer reached 70–80% confluence. For the PBDE transport experiments, the cells at passages 10–30 were seeded at 2×105 cells/cm2 into a 12well Transwell insert, and then cultured for 21 days. The culture medium was changed every 2 days. The Caco-2 cell monolayer was used to carry out the PBDE transepithelial transport experiment when the transepithelial electrical resistance (TEER) values were larger than 400 Ω/cm2 measured with an epithelial voltammeter (EVOM2, World Precision Instruments, Sarasota, FL, USA).
The methods used for sample extraction and cleanup were similar to those reported in our previous study [55]. After spiking with the surrogate standard 13C-PCB141, solutions from the AP and BL sides were extracted three times with acetone and a mixed solution of nhexane and dichloromethane. The obtained organic solutions were passed through a silica-alumina column to remove impurities. The mixed solvent of dichloromethane and n-hexane (v/v=1:1) was used as the mobile phase. The eluents containing PBDEs were collected and concentrated. Finally, the internal standard 13C-PCB208 was added. The samples were stored in 50 μL n-octane at −20 °C until GC/MS analysis. For PBDEs in the cells, following ultrasonic decomposition of the cells, the mixtures including cells were extracted and cleaned up as mentioned above.
2.2. MTT test
The PBDE concentrations were determined using a HewlettPackard (HP) 6890N gas chromatograph coupled with a 5975 mass spectrometer. Negative chemical ionization mode was used. Splitless injection of a 1-μL sample was performed. The temperature of the injector and ion source were set at 280 and 250 °C, respectively. Quantification of tri- to hepta-BDE congeners was achieved using a DB5MS capillary column (12 m×0.25 mm×0.1 µm, J & W Scientific, USA) with no interfering peaks. The selective ion monitoring mode was selected, and the ions m/z=79/81 were monitored for tri- to heptaBDE congeners, 476/478 for 13C-PCB208, and 372/374 for 13CPCB141.
chemicals. Several studies have investigated the transport ratios of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals to assess their bioavailability using the Caco2 cell monolayer. It was also reported that PAHs can affect the gene expression of enzymes and ABC transporters in Caco-2 cells [26]. In addition, it was reported that the transport of PAHs is mediated by BCRP and the aryl hydrocarbon receptor [19,31]. Organic cation transporters (OCTs), a type of influx transporter, are expressed in Caco-2 cells. These can mediate the transport of xenobiotics with relatively low molecular weights and hydrophilic organic cations with diverse molecular structures [24]. However, research on the transepithelial transport mechanism of environmental pollutants and investigations of the roles of the influx and efflux transporters during transport are limited, although there have been many studies on the transepithelial transport mechanism of drugs using the Caco-2 monolayer model. If there is a clear understanding of the transepithelial transport mechanism of pollutants in the human intestine, those chemicals with low bioavailability and toxicity may receive more attention for application in industrial products. It is necessary to understand the roles the transporters play in the transport process. The present study aimed to determine whether the transporters play an important role during the transport of PBDEs. Therefore, the main objective of this study was to investigate the transepithelial transport mechanism of PBDEs, and study the roles of the efflux transporters (P-gp, MRP, and BCRP) and the influx transporters (OCTs) during the transport process using a Caco-2 cell monolayer.
2.5. Instrumental analysis
Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl) −2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the Caco-2 cells were seeded in 96-well plastic plates at the density of 3000 per well with 100 μL medium, and cultured for 24 h. Then the cells were exposed to targets (DMSO or PBDEs) at given concentrations for 12 h. After the incubation, the medium containing targets was removed, and 100 μL of 0.25% mg·mL−1 MTT in medium culture was added. The plates were incubated at 37 °C in a 5% CO2 atmosphere for 4 h. Then, the culture medium was removed, and DMSO (150 μL) was added into each well to dissolve the dark blue crystal. The absorbance was measured at 490 nm using an iMark plate reader (BIO-RAD, USA). The experiments were repeated six times.
2.6. Quality assurance/quality control For each batch of seven samples, a procedural blank was processed to monitor interfering peaks. BDE209 was observed in some of the blank samples. Therefore, BDE209 is not discussed in the present study. Each experiment was carried out three to five times. Calibration
2.3. Transepithelial transport experiment In this experiment, the solvent nonane in the PBDE stock solution 94
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plots for all of the congeners had satisfactory linear regression coefficients (R2 > 0.99). The reported concentrations were not corrected against the recovery rates of the surrogate standard 13C-PCB141 (83.9 ± 11.5%).
3.0
2.7. Statistical analysis
2.0
Transported
A
Accumulated
PBDEs (ng)
2.5
The calculation equations of the present study were given in the Supporting Information. Statistical significance was analyzed by independent sample test or bivariate correlations unless otherwise specified. Relationships between two series of data were analyzed by linear regression. A correlation was considered statistically significant when p < 0.05.
y = 0.1234x + 0.0361 2
R = 0.999
1.5
y = 0.041x - 0.0171 2
R = 0.999
1.0 0.5
3. Results and discussion
0.0 0
3.1. Caco-2 cell viability
5
10
15
20
25
PBDE concentrations (ng/mL)
MTT test was used to evaluate cell survival in terms of mitochondrial viability. The cytotoxicity of DMSO and PBDEs in different concentrations to the Caco-2 cells was tested with MTT assay. Only concentrations above 80% viability were acceptable for the further study. The data shows that DMSO below the concentration of 0.2% and the concentrations of PBDEs even up to 20 ng/mL had no significant effects on the viability of Caco-2 cells (Fig. S1).
40% Transported
B
Accumulated
35% 2
y = -0.0294x + 0.5079x - 1.8564
30%
2
R = 0.896 Ratios
25%
3.2. Influence of PBDE concentrations on transepithelial transport
20% 15%
Analysis of concentration-dependent transepithelial transport is one of the methods used to investigate the transport mechanisms of chemicals, as it allows differentiation between carrier-mediated transport and passive transport [46]. In the present study, the transepithelial transport of PBDEs in the AP-to-BL direction at different concentrations for an exposure time of 12 h in the Caco-2 cell monolayer model was investigated. The highest concentration of 20 ng/mL was selected according to the solubility of the medium, then the 1/2 concentration was used for the gradient. The data are shown in Supporting Information (Table S1). Due to the similar characteristics of the PBDE congeners, the data averaged tri- to hepta-BDE congeners are shown in Fig. 1A. The transepithelial transport of the averaged tri- to hepta-BDEs varied widely from 0.086 to 0.799 ng when their concentrations increased from 2.5 to 20 ng/mL. At these concentrations, the transepithelial transport of PBDEs from the AP to BL chamber increased linearly (R2=0.999, p < 0.001). A similar tendency was observed for intracellular accumulation (Fig. 1A). A greater proportion of PBDEs accumulated in the cells and accounted for up to 37.6% (BDE190) of the total. In the present study, no saturation was observed even when the PBDE concentration reached 20 ng/mL for both the accumulated and transported PBDEs, which was consistent with a non-saturable mechanism. This mechanism indicated that passive diffusion dominated the transport. Therefore, these results demonstrated that PBDEs exhibited concentration-dependent accumulation and transport in the Caco-2 cell monolayer, suggesting that passive diffusion dominated the transepithelial transport of PBDEs at the concentration range tested [51]. According to the literature, the simple passive diffusion rate improved with increased concentration and the transport rate of the drug carried by transporters was almost constant [18,36,37]. The transport behavior of carrier-mediated uptake was consistent with a saturated mechanism at high concentrations [23]. On the one hand, high passive diffusion may overwhelm the transporter effect at high concentration, and on the other hand, the poor solubility of PBDEs might lead to a low concentration below the saturation level of transporters. The roles of transporters cannot be excluded by only
y = -0.0396x + 0.3643 10%
2
R = 0.906
5% 0% 5
6
7
8
9
LogK OW Fig. 1. The concentration effects on the transepithelial transport and intracellular accumulation of PBDEs (A: the relationship between the transported or accumulated mass and the concentrations of average PBDE congeners; B: the relationship between the transported or accumulated ratios and the LogKOW of PBDEs with the error bar of the data at different concentrations).
considering the influence of concentration. Thus, we also investigated whether the transporters were involved in the transport mechanism of PBDEs. Due to the different hydrophilicity and molecular volumes of the congeners with various bromine atoms, the transepithelial transport of individual PBDE congeners were different. Overall, lower brominated congeners tend to have higher permeation values compared to higher brominated congeners (Fig. 1B). There was a negative linear relationship between the permeation value and the LogKOW value for each congener. This demonstrated that higher brominated congeners are difficult to transport. The low solubility of higher brominated congeners may also have contributed to the low permeation values. However, a parabolic curve was observed between the level of intracellular accumulation and LogKOW (Fig. 1B). According to the parabolic equations between the accumulation ratios and LogKOW, the largest accumulation ratio was observed at the LogKOW of 8.64, which was higher than the LogKOW values of the target congeners. Accumulation of the higher brominated congeners in the cells was generally easier. This phenomenon was the opposite to that of the permeation values. This may be due to the higher hydrophilicity of higher brominated congeners which results in their accumulation as
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50% Transported
45%
to have higher permeation values compared to higher brominated congeners. The Papp(AP-BL) values obtained in the present study were between 2.34×10−7 and 8.39×10−7 cm/s. In the literature, chemicals with Papp values lower than 10×10−7 cm/s are classified as poorly absorbed compounds [53]. The present results indicated that the target PBDE congeners were all poorly absorbed compounds. In the opposite direction, PBDEs had Papp(BL-AP) values ranging from 6.53×10−8 to 1.04×10−6 cm/s, and the values decreased with increasing numbers of bromine atoms in the congeners. In studies of the transepithelial transport of drugs, the efflux ratio is usually used to investigate whether transporters participate in transport [9,28]. It is considered that transport has directionality when the ratio is larger than 1.5, and transport is not directional when the ratio is below this value. The present efflux ratios were all lower than 1.5 (Table S5), suggesting that the transport was not directional and indicated that passive diffusion may play an important role in the transport mechanism of PBDEs on the basis of the efflux ratios of the congeners. This is consistent with the concentration effect.
A
Accumulated
40%
Ratios
35% y = 0.0142x + 0.0988
30%
2
R = 0.921
25% 20% 15% 10%
y = 0.005x + 0.0128
5%
2
R = 0.976
0% 0
5
10
15
20
25
30
Time (h) 35% Transported
B
Accumulated
30%
3.4. Influence of temperature on transepithelial transport To investigate the presence of an energy-dependent transport mechanism, the effect of temperature on the transcellular transport of PBDEs was studied by incubating cells at 4 °C and at 37 °C (Fig. 3). The transport ratios of PBDEs across the Caco-2 cell monolayer in the AP-to-BL direction at 4 °C were all below 5.4%, and only a small proportion of PBDEs with a range from 0.3–2.4% was transported from the BL to the AP side (Fig. 3A). The number of transported PBDEs was lower at 4 °C than at 37 °C in both directions, although the data showed no statistically significant difference in the BL-to-AP direction. The cell accumulation ratios of PBDEs at 4 °C from the AP-to-BL side ranged between 5.6 and 35.4%, and between 0.2 and 2.6% for the BL-to-AP direction (Fig. 3B). For the BL-to-AP direction, the intracellular accumulation of PBDEs was generally lower at 4 °C, especially for BDE17, 183 and 190. However, for the AP-to-BL direction, the lower brominated congeners generally showed different results compared to penta- to hepta-congeners. This may be attributed to high transportation of PBDEs with lower brominated congeners. Rocha et al. [38] reported that a decrease in temperature reduced cellular metabolism and acted as an inhibitor of the energy-dependent transport mechanism. In the present study, low temperature significantly reduced the transport and accumulation of PBDEs, suggesting that an energy-dependent mechanism was involved in the bidirectional transport of PBDEs. This dependence on temperature may indicate the participation of active transport [38]. However, it should be noted that the decrease in temperature may also affect passive diffusion [46].
2
y = -0.0264x + 0.4516x - 1.6486
25%
2
Ratios
R = 0.875 20% 15% y = -0.0212x + 0.2096
10%
2
R = 0.874 5% 0% 5
6
7
8
9
LogK OW Fig. 2. Time effects on the transepithelial transport and intracellular accumulation of PBDEs for apical-to-basolateral direction (A: the relationship between the transported or accumulated ratios and the exposure time of average PBDE congeners; B: the relationship between the transported or accumulated ratios of average PBDE congeners during different exposure time and LogKOW).
cells generally contain many lipids. 3.3. Influence of time on transepithelial transport The transport of PBDEs across the Caco-2 cell monolayer from the AP to the BL chamber and in the opposite direction from 1 to 24 h was investigated (Table S2 and S3). As shown in Fig. 2A, the transepithelial transport ratios of averaged tri- to hepta-BDEs from the AP to BL side increased from 0.9–12.7% over time. Similar results were also observed in the opposite direction, but were less than 6.7% even after exposure for 24 h (Fig. S2A). Cellular accumulation of PBDEs in both directions increased with increased exposure time (Fig. 2A and S2A), and no saturation was observed. Several equations can be successfully used to describe the sorption kinetics of compounds on an adsorbent or a reaction process. In the present study, to determine the transepithelial transport and intracellular accumulation kinetics of PBDEs in the Caco-2 cell monolayer, the pseudo-first-order equation was used to fit the data (Table S4). The results showed that there were significant relationships, especially for transepithelial transport, which indicated the applicability of the pseudo-first-order model to describe the transepithelial transport and intracellular accumulation processes of PBDEs in the Caco-2 cell monolayer. The Papp values of PBDE congeners were calculated in both directions (Table S5). In general, lower brominated congeners tended
3.5. Influence of pH value on transepithelial transport To investigate whether hydrogen ion involves the transepithelial transport, the effect of pH on the transport of PBDEs were study. Caco2 cells were incubated at pH 5.5 and 7.4 in the AP chamber, and 7.4 in the BL chamber. As shown in Fig. 4A, the transport ratios at pH 5.5 were significantly lower than those at pH 7.4 for tri- to penta-BDE congeners, with the exception of BDE47. In addition, a decrease in the transport ratios of these chemicals was observed in the BL-to-AP direction. There were no obvious differences in transport of the hexaand hepta-BDE congeners in both directions. However, a slightly higher accumulation of PBDEs in the cells was observed at pH 5.5 in the AP-to-BL direction, especially for hexa- to hepta-BDE congeners (Fig. 4B). These results suggested that acid conditions in the AP medium improved the accumulation of PBDEs in cells, but may prevent transport through the Caco-2 cells. The present results were consistent with those observed by Kimura et al. [22] who found that the transcellular transport of domoic acid from the AP-to-BL side was optimal at neutral pH. The lower pH value 96
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16% AP-BL37
AP-BL4
BL-AP37
BL-AP4
A
14% Transported ratios
12% 10% 8% **
6%
**
4%
** **
**
2%
**
** **
**
**
**
*
** ** *
**
**
**
**
0% BDE17 BDE28 BDE71 BDE47 BDE66 BDE100 BDE99 BDE85 BDE154 BDE153 BDE138 BDE183 BDE190
50% AP-BL37
AP-BL4
BL-AP37
B
BL-AP4
**
**
**
30%
**
20%
**
**
**
**
10%
**
**
E1 38
E1 83
E1 90
**
BD
BD
BD
BD
**
E1 53
**
E1 54
E9 9 BD
**
E8 5
**
BD
**
BD
BD
BD
**
E1 00
E4 7
E7 1
BD
BD
E2 8
E1 7
0%
BD
**
**
E6 6
**
BD
Accumulated ratios
40%
Fig. 3. Temperature effects on the transepithelial transport and intracellular accumulation of PBDEs (Comparison of the results at 4 °C with 37 °C; **p < 0.01; *p < 0.05).
assumed that the isorhamnetin forms might account for the changes in its directional transport through cell membranes. As reported in the literature, passive diffusion can be affected by the form of compounds present at different pH values [25]. The molecular form seems to be transported more easily. The present data were consistent with this theory. PBDEs in water may exist mainly in the form of particulates, and the dissolved/water partition ratio was related to their LogKOW [5].
of 5.5 led to lower transport of the chemical. Similar results were also reported for other chemicals, such as phenylethylamine [14], and isorhamnetin [12], although opposing results showed higher transport of these drugs at lower pH value. These findings may be attributed to the different physical and chemical properties of these chemicals. For example, the form (i.e., ionized or molecular form) of a chemical in solution is very important. In the report by Duan et al. [12], it was
16% AP-BL7.4
14%
AP-BL5.5
BL-AP7.4
A
BL-AP5.5
Transported ratios
**
12% 10% 8%
**
**
6%
**
4%
** *
2%
**
** *
**
*
*
*
*
BD
E1 90 BD
E1 83
E1 38 BD
E1 53 BD
E1 54 BD
BD
E8 5
E9 9 BD
E1 00 BD
BD
E6 6
E4 7 BD
BD
BD
BD
E1 7
E2 8
E7 1
0%
50%
Accumulated ratios
AP-BL7.4
AP-BL5.5
BL-AP7.4
40%
BL-AP5.5 *
B
*
*
*
*
**
**
30% 20% 10% *
*
*
*
E1 90 BD
E1 83 BD
E1 38 BD
E1 53 BD
E1 54 BD
E8 5 BD
E9 9 BD
E1 00 BD
E6 6 BD
E4 7 BD
E7 1 BD
E2 8 BD
BD
E1 7
0%
Fig. 4. The effects of pH on the transepithelial transport and intracellular accumulation of PBDEs (Comparison of the results at pH 5.5 with 7.4; **p < 0.01; *p < 0.05).
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varied between 1.0 and 5.0% (Fig. S3). Compared with the control, the intracellular accumulation ratios increased significantly for all congeners in both directions when verapamil was added, while the transport ratios decreased slightly. Similar results were observed for the other two efflux transporters (Fig. 5 and S3). The presence of MK571, an MRP inhibitor, did not significantly affect transepithelial transport. However, this inhibitor significantly affected the intracellular accumulation of AP-to-BL transport. For the BL-to-AP direction, addition of the inhibitor did not significantly affect the transepithelial transport and intracellular accumulation ratios of most congeners. For BCRP, the transport of PBDEs from the BL-to-AP side decreased when Ko143 was added, and statistically significant differences were observed for most congeners. The intracellular accumulation of all congeners significantly increased. For the BL-to-AP direction, there were no significant differences compared with the control for both the transport and accumulation ratios (Fig. S3). Considering the low expression of BCRP in Caco-2 cells, the effect of BCRP on the transport of PBDEs in the human intestine may be underestimated [47]. Caco-2 cells, derived from human colonic adenocarcinoma, express many functional transporters and have been widely used to study the transport processes of xenobiotics [15,16,32,40,49]. P-gp, MRP, and BCRP are functionally expressed in the Caco-2 cell monolayer and can be applied to a wide range of substrates [8,50]. The transport of P-gp substrates increased in the AP-to-BL direction and decreased in the BL-to-AP direction in the presence of inhibitors. Although it seemed that the efflux transporters were not involved in the transport from the transepithelial transport ratios of PBDEs in both directions, the significantly higher intracellular accumulation ratios may indicate the actions of the efflux transporters after the inhibitors were added. These efflux carriers decreased the absorption of administered xenobiotics by preventing their intracellular accumulation and the initial step in the transport of P-gp substrates due to their interactions with the bilayer lipid members [45]. It was reported that biosorption played an important role in the biodegradation of PAHs and that trans membrane transport is the key determinant for further intracellular biochemical processes [4,11]. Intracellular accumulations were significantly in-
The pH value of the solution might affect both the existing forms and the charge species of PBDEs. Oxygen atom in PBDEs may have a charge of H+ in acid conditions due to the high electronegativity of the atom. Thus, PBDEs in acid solution may have a charge of H+. It seems that the molecular form of PBDEs is easily transported compared with the H+ charged PBDEs. Therefore, we found that the low brominated BDEs had higher transport ratios at pH 7.4 than those at pH 5.5. However, it should be noted that because transport is the result of many factors, such as hydrophobicity, molecular volume, and transporters, this resulted in the high brominated BDEs not showing significantly higher transport ratios at pH 7.4 than those at pH 5.5. For example, organic anion transporting polypeptides (OATPs), highly expressed transporters in mouse liver (also expressed in Caco-2 cells), can transport BDE47, BDE99, and BDE153 [33,34,42]. In addition, human organic anion transporting polypeptide B (OATP2B1) is a pH-sensitive transporter and is expressed in the AP membranes of small intestinal epithelial cells [39]. If these transporters are involved in PBDE transport, more information should be obtained to understand the influence of pH. 3.6. Influence of efflux transporters on transepithelial transport As previously mentioned, the involvement of efflux transporters in the transport of PBDEs cannot be determined based on the influence of concentration alone. To understand the involvement of these transporters, we investigated the roles of three efflux transporters (P-gp, MRP, and BCRP) generally used to study the efflux transport of drugs using the Caco-2 cell monolayer. Three inhibitors, verapamil, MK571 and Ko143, were used to inhibit the expression of the three transporters, respectively, and were added to the chambers. The transport and intracellular accumulation ratios with or without the inhibitors were determined in both directions. The results are shown in Fig. 5 and S3. The transport ratios of PBDEs in the AP-to-BL direction ranged between 0.5 and 15.5% (Fig. 5A), and the intracellular accumulation ratios were between 26.9 and 58.1% when verapamil, a P-gp inhibitor, was added (Fig. 5B). For the BL-to-AP direction, the transport ratios ranged from 0.2 to 4.9%, and the intracellular accumulation ratios 20% P-gp
Transported atios
15%
MRP
BCRP
OCT
A
Control
* ** *
10% *
* *
** **
**
*
5%
*
* * ***
**
**
*
**
** *
*
**
**
** **
**
**
**
* **
0% BDE17
BDE28
BDE71
BDE47
MRP
BCRP
OCT
BDE66
BDE100
BDE99
BDE85
BDE154 BDE153 BDE138 BDE183 BDE190
80% P-gp
B
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Accumulated ratios
70% 60% 50% 40% **
30% 20%
**
**
** **
**
** **
**
** **
**
**
10% 0% BDE17
BDE28
BDE71
BDE47
BDE66
BDE100
BDE99
BDE85
BDE154 BDE153 BDE138 BDE183 BDE190
Fig. 5. The effects of efflux and influx transporters on the transepithelial transport and intracellular accumulation of PBDEs (**p < 0.01; *p < 0.05).
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they have low permeability [35]. The present results were consistent with this rule. In the present study, the molecular weights of tri- to penta-BDEs were 406.7–564.5 g/mol, which were lower than those of hexa- (643.4 g/mol) and hepta-BDEs (722.3 g/mol). The present results may indicate that OCTs tend to participate in the transport of tri- to penta-BDEs, while transport of higher brominated congeners is limited. However, further research is required to understand the accumulation of PBDEs in cells after the addition of cimetidine. To further confirm the involvement of the aforementioned transporters during transepithelial transport, further investigations are necessary. For example, the use of gene transfection techniques to establish cell lines with expression of the target genes (the transporters), and subsequent transepithelial transport experiments. 4. Conclusions In the present study, the transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine was determined using a Caco-2 cell monolayer. The results of the present study were showed in Fig. 6. These results suggest the following: (1) passive diffusion dominated the transepithelial transport of PBDEs in the concentration ranges used in this experiment, and transepithelial transport of PBDEs follows pseudo-first-order kinetics; (2) the ratedetermining step of the transepithelial transport of PBDEs is trans-cell transport including the trans-pore process, and the Caco-2 cell monolayer might not be suitable for estimating the bioavailability of chemicals with high LogKOW values; (3) a pH-dependent transport mechanism was involved; (4) the efflux transporters P-gp, MRP, and BCRP might participate in the transepithelial transport of PBDEs; (5) the influx transporters, OCTs, seemed to participate in the transport of tri- to penta-BDEs, while the transport of higher brominated congeners was limited.
Fig. 6. The possible transepithelial transport mechanisms of PBDEs in Caco-2 cell monolayer.
creased in the AP-to-BL direction in the present study. This may be because addition of the inhibitors prevented PBDE efflux from the cells to the AP chamber and caused increased intracellular accumulation. According to the analysis previously mentioned, we assume that PBDEs adsorption onto Caco-2 cells is a rapid process, while the process of entering the cell membrane is slow. The rate-determining step in the transepithelial transport of PBDEs in the Caco-2 cell monolayer is the trans-cell transport, i.e., the transport of PBDEs from the intramembrane of the AP-to-BL side, and vice versa. In addition, considering the very different intracellular accumulation and transepithelial transport ratios between the AP-to-BL and BLto-AP directions, we assumed that the pore of the Transwell insert also had an important effect on the transport ratios of PBDEs. The average membrane pore area in the Transwell insert accounted for less than 0.37% (9 images using three Transwell inserts) of the total area calculated using Nano Measurer software after scanning the surface topography using JSM-7500F (JEOL) scanning electron microscopy (Fig. S4). Thus, we assumed that the rate-determining step of the apparent transepithelial transport ratios may include the trans-pore process (Fig. 6). To estimate the bioavailability of chemicals, both the intracellular accumulation and transepithelial transport of chemicals should be considered [15]. However, we assumed that PBDEs were mainly adsorbed in the phospholipid membrane of the AP side, especially the higher brominated congeners due to high lipophilicity. Actually, the transport of these chemicals was lower. Therefore, we assumed that the Caco-2 cell monolayer might not be suitable for estimating the bioavailability of chemicals with high LogKOW values, such as PBDEs.
Notes The authors declare no competing financial interest. Acknowledgements This research was financially supported by the National Nature Science Foundation of China (Nos. 21277086 and 21677094). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.envres.2016.12.024. References
3.7. Influence of influx transporter on transepithelial transport
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An inhibition assay was carried out in the AP-to-BL direction by adding 50 μM cimetidine, an inhibitor of OCTs (Fig. 5). The transport ratios of tri- to penta-BDEs significantly decreased, which indicated that OCTs may participate in the transport of lower brominated BDEs. In addition, the intracellular accumulation ratios significantly increased compared with the control. OCTs are expressed in the human small intestine. They mediate the transport of some organic compounds including tetraethyl ammonium, acetylcholine, choline, and procainamide [24,48]. In intestinal epithelia, OCTs are present in the lateral membrane (OCT1) and the brush border membrane (OCT3) [13,30,42]. It was reported that OCTs tend to affect the transport of compounds with lower molecular weight, higher hydrophilicity and positively charged compounds [17]. In addition, according to the Rule of Five, chemicals with a molecular weight larger than 500 g/mol are not generally suitable for drugs, as 99
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The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer
MARK
⁎
Yingxin Yu , Mengmeng Wang, Kaiqiong Zhang, Dan Yang, Yufang Zhong, Jing An, Bingli Lei, Xinyu Zhang Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China
A R T I C L E I N F O
A BS T RAC T
Keywords: Caco-2 cell monolayer Efflux transporter Influx transporter Polybrominated biphenyl ethers Transepithelial transport
Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH.
1. Introduction Polybrominated diphenyl ethers (PBDEs) are a class of typical persistent organic pollutants (POPs), which can cause adverse effects in the ecosystem and in humans. They were widely used as brominated flame retardants in various consumer products, especially in textiles, plastics, and electronic appliances [10]. As they are not chemically bound to the materials, they can be released from the materials into the environment. Due to their stability and semi-volatility, PBDEs are difficult to degrade in the environment and can be transported long distances [21]. In addition to their high lipophilicity, PBDEs are persistent with the potential for bioaccumulation in organisms and biomagnification through food chains. They have been detected in all types of biota [1,20,29,54]. In the past few years, the levels of PBDEs in biota have increased significantly and PBDEs have been detected in human tissue samples [1,20,41,43]. Toxicity studies have shown that PBDEs result in embryotoxicity, developmental neurotoxicity, and
reproductive toxicity [10,3,7]. The risks due to these chemicals have attracted increasing attention in recent years. Oral ingestion is the main exposure pathway for PBDEs in the non-professional population [27]. Caco-2 cells, derived from human colon carcinoma, express tight junctions, micro villi, and many enzymes and transporters present in the normal human small intestine [40]. These cells can differentiate and polarize, and form a monolayer when cultured under specific conditions. Because of their similar morphology and function to human small intestinal epithelial cells, the Caco-2 cell model is widely used as a tool to investigate the absorption, transport, and metabolism of xenobiotics [15,16,32,44,49]. The efflux transporters, P-glycoprotein (p-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP), which belong to the ATP-binding cassette transporters (ABC transporters), have been shown to pump drugs back to the lumen, and act as a biological barrier, which can affect the bioavailability of
Abbreviations: ABC transporters, ATP-binding cassette transporters; AP, apical; BCRP, breast cancer resistance protein; BL, basolateral; DMSO, dimethyl sulphoxide; EDTA, ethylenediamine tetraacetic acid; GC/MS, gas chromatography/mass spectrometry; MEM, minimum essential medium; MRP, multidrug resistance-associated protein; OATP2B1, organic anion transporting polypeptide B; OATPs, organic anion transporting polypeptides; OCTs, Organic cation transporters; PAHs, polycyclic aromatic hydrocarbons; PBDEs, polybrominated diphenyl ethers; PCBs, polychlorinated biphenyls; P-gp, P-glycoprotein; POPs, persistent organic pollutants; TEER, transepithelial electrical resistance. ⁎ Corresponding author. E-mail address: yuyingxin@staff.shu.edu.cn (Y. Yu). http://dx.doi.org/10.1016/j.envres.2016.12.024 Received 29 August 2016; Received in revised form 2 December 2016; Accepted 20 December 2016 0013-9351/ © 2016 Elsevier Inc. All rights reserved.
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2. Material and methods
was changed to dimethyl sulphoxide (DMSO). Prior to PBDE exposure, the Caco-2 cell monolayer was washed three times with D-Hanks solution. After pre-incubation, the buffer was then gently removed using nitrogen. To investigate the influence of time on transepithelial transport, PBDEs were added into the apical (AP) side or basolateral (BL) side. The volume of medium added to the AP and BL chambers was 0.5 and 1.5 mL, respectively. Following incubation, the solutions from the AP and BL sides were collected and the cells were harvested using 0.25% trypsin and 0.03% EDTA, respectively. PBDEs in the solutions from the AP and BL sides were extracted and determined using gas chromatography/mass spectrometry (GC/MS). For PBDEs in the cells, the harvested cells were disrupted using cell breaking apparatus, and then extracted as described previously. To investigate the influence of PBDE concentrations on transport, four concentrations of PBDE (2.5, 5, 10, and 20 ng/mL) in the medium were used. Various PBDE exposure times (1, 2, 4, 8, 12, and 24 h) at the PBDE concentration of 10 ng/mL were used to study the time effects. In subsequent experiments, the PBDE concentration was set at 10 ng/mL, and the PBDE exposure time was 12 h. The influence of pH on the transport of PBDEs in both directions, i.e., AP to BL and BL to AP, was determined using the following paired pH values in the AP/BL compartments, i.e., 5.5/7.4 and 7.4/7.4. The experiment was carried out at two temperatures (37 and 4 °C) to investigate the influence of energy. To determine the roles of P-gp, MRP, BCRP, and OCTs during transport, 100 μM verapamil [6], 100 μM MK571 [52], 10 μM Ko143 [12], and 50 μM cimetidine [2] were used as inhibitors, respectively. We found that concentrations of DMSO below 0.2% had no significant effect on the viability of Caco-2 cells (data not shown). Therefore the concentrations of DMSO used in the present study were below 0.2%.
2.1. Caco-2 cell culture
2.4. Analytical protocol
Caco-2 cells, purchased from BioHermes Bio & Medical Technology Co., Inc. (Wuxi, China), were cultured in minimum essential medium (MEM) supplemented with 20% fetal bovine serum and 1% penicillin. The cells were grown in a 5% CO2 atmosphere at 37 °C under humidified conditions. The culture medium was replaced every 1–2 days. The cells were passaged using 0.25% trypsin and 0.03% ethylenediamine tetraacetic acid (EDTA) until the cell monolayer reached 70–80% confluence. For the PBDE transport experiments, the cells at passages 10–30 were seeded at 2×105 cells/cm2 into a 12well Transwell insert, and then cultured for 21 days. The culture medium was changed every 2 days. The Caco-2 cell monolayer was used to carry out the PBDE transepithelial transport experiment when the transepithelial electrical resistance (TEER) values were larger than 400 Ω/cm2 measured with an epithelial voltammeter (EVOM2, World Precision Instruments, Sarasota, FL, USA).
The methods used for sample extraction and cleanup were similar to those reported in our previous study [55]. After spiking with the surrogate standard 13C-PCB141, solutions from the AP and BL sides were extracted three times with acetone and a mixed solution of nhexane and dichloromethane. The obtained organic solutions were passed through a silica-alumina column to remove impurities. The mixed solvent of dichloromethane and n-hexane (v/v=1:1) was used as the mobile phase. The eluents containing PBDEs were collected and concentrated. Finally, the internal standard 13C-PCB208 was added. The samples were stored in 50 μL n-octane at −20 °C until GC/MS analysis. For PBDEs in the cells, following ultrasonic decomposition of the cells, the mixtures including cells were extracted and cleaned up as mentioned above.
2.2. MTT test
The PBDE concentrations were determined using a HewlettPackard (HP) 6890N gas chromatograph coupled with a 5975 mass spectrometer. Negative chemical ionization mode was used. Splitless injection of a 1-μL sample was performed. The temperature of the injector and ion source were set at 280 and 250 °C, respectively. Quantification of tri- to hepta-BDE congeners was achieved using a DB5MS capillary column (12 m×0.25 mm×0.1 µm, J & W Scientific, USA) with no interfering peaks. The selective ion monitoring mode was selected, and the ions m/z=79/81 were monitored for tri- to heptaBDE congeners, 476/478 for 13C-PCB208, and 372/374 for 13CPCB141.
chemicals. Several studies have investigated the transport ratios of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals to assess their bioavailability using the Caco2 cell monolayer. It was also reported that PAHs can affect the gene expression of enzymes and ABC transporters in Caco-2 cells [26]. In addition, it was reported that the transport of PAHs is mediated by BCRP and the aryl hydrocarbon receptor [19,31]. Organic cation transporters (OCTs), a type of influx transporter, are expressed in Caco-2 cells. These can mediate the transport of xenobiotics with relatively low molecular weights and hydrophilic organic cations with diverse molecular structures [24]. However, research on the transepithelial transport mechanism of environmental pollutants and investigations of the roles of the influx and efflux transporters during transport are limited, although there have been many studies on the transepithelial transport mechanism of drugs using the Caco-2 monolayer model. If there is a clear understanding of the transepithelial transport mechanism of pollutants in the human intestine, those chemicals with low bioavailability and toxicity may receive more attention for application in industrial products. It is necessary to understand the roles the transporters play in the transport process. The present study aimed to determine whether the transporters play an important role during the transport of PBDEs. Therefore, the main objective of this study was to investigate the transepithelial transport mechanism of PBDEs, and study the roles of the efflux transporters (P-gp, MRP, and BCRP) and the influx transporters (OCTs) during the transport process using a Caco-2 cell monolayer.
2.5. Instrumental analysis
Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl) −2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the Caco-2 cells were seeded in 96-well plastic plates at the density of 3000 per well with 100 μL medium, and cultured for 24 h. Then the cells were exposed to targets (DMSO or PBDEs) at given concentrations for 12 h. After the incubation, the medium containing targets was removed, and 100 μL of 0.25% mg·mL−1 MTT in medium culture was added. The plates were incubated at 37 °C in a 5% CO2 atmosphere for 4 h. Then, the culture medium was removed, and DMSO (150 μL) was added into each well to dissolve the dark blue crystal. The absorbance was measured at 490 nm using an iMark plate reader (BIO-RAD, USA). The experiments were repeated six times.
2.6. Quality assurance/quality control For each batch of seven samples, a procedural blank was processed to monitor interfering peaks. BDE209 was observed in some of the blank samples. Therefore, BDE209 is not discussed in the present study. Each experiment was carried out three to five times. Calibration
2.3. Transepithelial transport experiment In this experiment, the solvent nonane in the PBDE stock solution 94
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plots for all of the congeners had satisfactory linear regression coefficients (R2 > 0.99). The reported concentrations were not corrected against the recovery rates of the surrogate standard 13C-PCB141 (83.9 ± 11.5%).
3.0
2.7. Statistical analysis
2.0
Transported
A
Accumulated
PBDEs (ng)
2.5
The calculation equations of the present study were given in the Supporting Information. Statistical significance was analyzed by independent sample test or bivariate correlations unless otherwise specified. Relationships between two series of data were analyzed by linear regression. A correlation was considered statistically significant when p < 0.05.
y = 0.1234x + 0.0361 2
R = 0.999
1.5
y = 0.041x - 0.0171 2
R = 0.999
1.0 0.5
3. Results and discussion
0.0 0
3.1. Caco-2 cell viability
5
10
15
20
25
PBDE concentrations (ng/mL)
MTT test was used to evaluate cell survival in terms of mitochondrial viability. The cytotoxicity of DMSO and PBDEs in different concentrations to the Caco-2 cells was tested with MTT assay. Only concentrations above 80% viability were acceptable for the further study. The data shows that DMSO below the concentration of 0.2% and the concentrations of PBDEs even up to 20 ng/mL had no significant effects on the viability of Caco-2 cells (Fig. S1).
40% Transported
B
Accumulated
35% 2
y = -0.0294x + 0.5079x - 1.8564
30%
2
R = 0.896 Ratios
25%
3.2. Influence of PBDE concentrations on transepithelial transport
20% 15%
Analysis of concentration-dependent transepithelial transport is one of the methods used to investigate the transport mechanisms of chemicals, as it allows differentiation between carrier-mediated transport and passive transport [46]. In the present study, the transepithelial transport of PBDEs in the AP-to-BL direction at different concentrations for an exposure time of 12 h in the Caco-2 cell monolayer model was investigated. The highest concentration of 20 ng/mL was selected according to the solubility of the medium, then the 1/2 concentration was used for the gradient. The data are shown in Supporting Information (Table S1). Due to the similar characteristics of the PBDE congeners, the data averaged tri- to hepta-BDE congeners are shown in Fig. 1A. The transepithelial transport of the averaged tri- to hepta-BDEs varied widely from 0.086 to 0.799 ng when their concentrations increased from 2.5 to 20 ng/mL. At these concentrations, the transepithelial transport of PBDEs from the AP to BL chamber increased linearly (R2=0.999, p < 0.001). A similar tendency was observed for intracellular accumulation (Fig. 1A). A greater proportion of PBDEs accumulated in the cells and accounted for up to 37.6% (BDE190) of the total. In the present study, no saturation was observed even when the PBDE concentration reached 20 ng/mL for both the accumulated and transported PBDEs, which was consistent with a non-saturable mechanism. This mechanism indicated that passive diffusion dominated the transport. Therefore, these results demonstrated that PBDEs exhibited concentration-dependent accumulation and transport in the Caco-2 cell monolayer, suggesting that passive diffusion dominated the transepithelial transport of PBDEs at the concentration range tested [51]. According to the literature, the simple passive diffusion rate improved with increased concentration and the transport rate of the drug carried by transporters was almost constant [18,36,37]. The transport behavior of carrier-mediated uptake was consistent with a saturated mechanism at high concentrations [23]. On the one hand, high passive diffusion may overwhelm the transporter effect at high concentration, and on the other hand, the poor solubility of PBDEs might lead to a low concentration below the saturation level of transporters. The roles of transporters cannot be excluded by only
y = -0.0396x + 0.3643 10%
2
R = 0.906
5% 0% 5
6
7
8
9
LogK OW Fig. 1. The concentration effects on the transepithelial transport and intracellular accumulation of PBDEs (A: the relationship between the transported or accumulated mass and the concentrations of average PBDE congeners; B: the relationship between the transported or accumulated ratios and the LogKOW of PBDEs with the error bar of the data at different concentrations).
considering the influence of concentration. Thus, we also investigated whether the transporters were involved in the transport mechanism of PBDEs. Due to the different hydrophilicity and molecular volumes of the congeners with various bromine atoms, the transepithelial transport of individual PBDE congeners were different. Overall, lower brominated congeners tend to have higher permeation values compared to higher brominated congeners (Fig. 1B). There was a negative linear relationship between the permeation value and the LogKOW value for each congener. This demonstrated that higher brominated congeners are difficult to transport. The low solubility of higher brominated congeners may also have contributed to the low permeation values. However, a parabolic curve was observed between the level of intracellular accumulation and LogKOW (Fig. 1B). According to the parabolic equations between the accumulation ratios and LogKOW, the largest accumulation ratio was observed at the LogKOW of 8.64, which was higher than the LogKOW values of the target congeners. Accumulation of the higher brominated congeners in the cells was generally easier. This phenomenon was the opposite to that of the permeation values. This may be due to the higher hydrophilicity of higher brominated congeners which results in their accumulation as
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50% Transported
45%
to have higher permeation values compared to higher brominated congeners. The Papp(AP-BL) values obtained in the present study were between 2.34×10−7 and 8.39×10−7 cm/s. In the literature, chemicals with Papp values lower than 10×10−7 cm/s are classified as poorly absorbed compounds [53]. The present results indicated that the target PBDE congeners were all poorly absorbed compounds. In the opposite direction, PBDEs had Papp(BL-AP) values ranging from 6.53×10−8 to 1.04×10−6 cm/s, and the values decreased with increasing numbers of bromine atoms in the congeners. In studies of the transepithelial transport of drugs, the efflux ratio is usually used to investigate whether transporters participate in transport [9,28]. It is considered that transport has directionality when the ratio is larger than 1.5, and transport is not directional when the ratio is below this value. The present efflux ratios were all lower than 1.5 (Table S5), suggesting that the transport was not directional and indicated that passive diffusion may play an important role in the transport mechanism of PBDEs on the basis of the efflux ratios of the congeners. This is consistent with the concentration effect.
A
Accumulated
40%
Ratios
35% y = 0.0142x + 0.0988
30%
2
R = 0.921
25% 20% 15% 10%
y = 0.005x + 0.0128
5%
2
R = 0.976
0% 0
5
10
15
20
25
30
Time (h) 35% Transported
B
Accumulated
30%
3.4. Influence of temperature on transepithelial transport To investigate the presence of an energy-dependent transport mechanism, the effect of temperature on the transcellular transport of PBDEs was studied by incubating cells at 4 °C and at 37 °C (Fig. 3). The transport ratios of PBDEs across the Caco-2 cell monolayer in the AP-to-BL direction at 4 °C were all below 5.4%, and only a small proportion of PBDEs with a range from 0.3–2.4% was transported from the BL to the AP side (Fig. 3A). The number of transported PBDEs was lower at 4 °C than at 37 °C in both directions, although the data showed no statistically significant difference in the BL-to-AP direction. The cell accumulation ratios of PBDEs at 4 °C from the AP-to-BL side ranged between 5.6 and 35.4%, and between 0.2 and 2.6% for the BL-to-AP direction (Fig. 3B). For the BL-to-AP direction, the intracellular accumulation of PBDEs was generally lower at 4 °C, especially for BDE17, 183 and 190. However, for the AP-to-BL direction, the lower brominated congeners generally showed different results compared to penta- to hepta-congeners. This may be attributed to high transportation of PBDEs with lower brominated congeners. Rocha et al. [38] reported that a decrease in temperature reduced cellular metabolism and acted as an inhibitor of the energy-dependent transport mechanism. In the present study, low temperature significantly reduced the transport and accumulation of PBDEs, suggesting that an energy-dependent mechanism was involved in the bidirectional transport of PBDEs. This dependence on temperature may indicate the participation of active transport [38]. However, it should be noted that the decrease in temperature may also affect passive diffusion [46].
2
y = -0.0264x + 0.4516x - 1.6486
25%
2
Ratios
R = 0.875 20% 15% y = -0.0212x + 0.2096
10%
2
R = 0.874 5% 0% 5
6
7
8
9
LogK OW Fig. 2. Time effects on the transepithelial transport and intracellular accumulation of PBDEs for apical-to-basolateral direction (A: the relationship between the transported or accumulated ratios and the exposure time of average PBDE congeners; B: the relationship between the transported or accumulated ratios of average PBDE congeners during different exposure time and LogKOW).
cells generally contain many lipids. 3.3. Influence of time on transepithelial transport The transport of PBDEs across the Caco-2 cell monolayer from the AP to the BL chamber and in the opposite direction from 1 to 24 h was investigated (Table S2 and S3). As shown in Fig. 2A, the transepithelial transport ratios of averaged tri- to hepta-BDEs from the AP to BL side increased from 0.9–12.7% over time. Similar results were also observed in the opposite direction, but were less than 6.7% even after exposure for 24 h (Fig. S2A). Cellular accumulation of PBDEs in both directions increased with increased exposure time (Fig. 2A and S2A), and no saturation was observed. Several equations can be successfully used to describe the sorption kinetics of compounds on an adsorbent or a reaction process. In the present study, to determine the transepithelial transport and intracellular accumulation kinetics of PBDEs in the Caco-2 cell monolayer, the pseudo-first-order equation was used to fit the data (Table S4). The results showed that there were significant relationships, especially for transepithelial transport, which indicated the applicability of the pseudo-first-order model to describe the transepithelial transport and intracellular accumulation processes of PBDEs in the Caco-2 cell monolayer. The Papp values of PBDE congeners were calculated in both directions (Table S5). In general, lower brominated congeners tended
3.5. Influence of pH value on transepithelial transport To investigate whether hydrogen ion involves the transepithelial transport, the effect of pH on the transport of PBDEs were study. Caco2 cells were incubated at pH 5.5 and 7.4 in the AP chamber, and 7.4 in the BL chamber. As shown in Fig. 4A, the transport ratios at pH 5.5 were significantly lower than those at pH 7.4 for tri- to penta-BDE congeners, with the exception of BDE47. In addition, a decrease in the transport ratios of these chemicals was observed in the BL-to-AP direction. There were no obvious differences in transport of the hexaand hepta-BDE congeners in both directions. However, a slightly higher accumulation of PBDEs in the cells was observed at pH 5.5 in the AP-to-BL direction, especially for hexa- to hepta-BDE congeners (Fig. 4B). These results suggested that acid conditions in the AP medium improved the accumulation of PBDEs in cells, but may prevent transport through the Caco-2 cells. The present results were consistent with those observed by Kimura et al. [22] who found that the transcellular transport of domoic acid from the AP-to-BL side was optimal at neutral pH. The lower pH value 96
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16% AP-BL37
AP-BL4
BL-AP37
BL-AP4
A
14% Transported ratios
12% 10% 8% **
6%
**
4%
** **
**
2%
**
** **
**
**
**
*
** ** *
**
**
**
**
0% BDE17 BDE28 BDE71 BDE47 BDE66 BDE100 BDE99 BDE85 BDE154 BDE153 BDE138 BDE183 BDE190
50% AP-BL37
AP-BL4
BL-AP37
B
BL-AP4
**
**
**
30%
**
20%
**
**
**
**
10%
**
**
E1 38
E1 83
E1 90
**
BD
BD
BD
BD
**
E1 53
**
E1 54
E9 9 BD
**
E8 5
**
BD
**
BD
BD
BD
**
E1 00
E4 7
E7 1
BD
BD
E2 8
E1 7
0%
BD
**
**
E6 6
**
BD
Accumulated ratios
40%
Fig. 3. Temperature effects on the transepithelial transport and intracellular accumulation of PBDEs (Comparison of the results at 4 °C with 37 °C; **p < 0.01; *p < 0.05).
assumed that the isorhamnetin forms might account for the changes in its directional transport through cell membranes. As reported in the literature, passive diffusion can be affected by the form of compounds present at different pH values [25]. The molecular form seems to be transported more easily. The present data were consistent with this theory. PBDEs in water may exist mainly in the form of particulates, and the dissolved/water partition ratio was related to their LogKOW [5].
of 5.5 led to lower transport of the chemical. Similar results were also reported for other chemicals, such as phenylethylamine [14], and isorhamnetin [12], although opposing results showed higher transport of these drugs at lower pH value. These findings may be attributed to the different physical and chemical properties of these chemicals. For example, the form (i.e., ionized or molecular form) of a chemical in solution is very important. In the report by Duan et al. [12], it was
16% AP-BL7.4
14%
AP-BL5.5
BL-AP7.4
A
BL-AP5.5
Transported ratios
**
12% 10% 8%
**
**
6%
**
4%
** *
2%
**
** *
**
*
*
*
*
BD
E1 90 BD
E1 83
E1 38 BD
E1 53 BD
E1 54 BD
BD
E8 5
E9 9 BD
E1 00 BD
BD
E6 6
E4 7 BD
BD
BD
BD
E1 7
E2 8
E7 1
0%
50%
Accumulated ratios
AP-BL7.4
AP-BL5.5
BL-AP7.4
40%
BL-AP5.5 *
B
*
*
*
*
**
**
30% 20% 10% *
*
*
*
E1 90 BD
E1 83 BD
E1 38 BD
E1 53 BD
E1 54 BD
E8 5 BD
E9 9 BD
E1 00 BD
E6 6 BD
E4 7 BD
E7 1 BD
E2 8 BD
BD
E1 7
0%
Fig. 4. The effects of pH on the transepithelial transport and intracellular accumulation of PBDEs (Comparison of the results at pH 5.5 with 7.4; **p < 0.01; *p < 0.05).
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varied between 1.0 and 5.0% (Fig. S3). Compared with the control, the intracellular accumulation ratios increased significantly for all congeners in both directions when verapamil was added, while the transport ratios decreased slightly. Similar results were observed for the other two efflux transporters (Fig. 5 and S3). The presence of MK571, an MRP inhibitor, did not significantly affect transepithelial transport. However, this inhibitor significantly affected the intracellular accumulation of AP-to-BL transport. For the BL-to-AP direction, addition of the inhibitor did not significantly affect the transepithelial transport and intracellular accumulation ratios of most congeners. For BCRP, the transport of PBDEs from the BL-to-AP side decreased when Ko143 was added, and statistically significant differences were observed for most congeners. The intracellular accumulation of all congeners significantly increased. For the BL-to-AP direction, there were no significant differences compared with the control for both the transport and accumulation ratios (Fig. S3). Considering the low expression of BCRP in Caco-2 cells, the effect of BCRP on the transport of PBDEs in the human intestine may be underestimated [47]. Caco-2 cells, derived from human colonic adenocarcinoma, express many functional transporters and have been widely used to study the transport processes of xenobiotics [15,16,32,40,49]. P-gp, MRP, and BCRP are functionally expressed in the Caco-2 cell monolayer and can be applied to a wide range of substrates [8,50]. The transport of P-gp substrates increased in the AP-to-BL direction and decreased in the BL-to-AP direction in the presence of inhibitors. Although it seemed that the efflux transporters were not involved in the transport from the transepithelial transport ratios of PBDEs in both directions, the significantly higher intracellular accumulation ratios may indicate the actions of the efflux transporters after the inhibitors were added. These efflux carriers decreased the absorption of administered xenobiotics by preventing their intracellular accumulation and the initial step in the transport of P-gp substrates due to their interactions with the bilayer lipid members [45]. It was reported that biosorption played an important role in the biodegradation of PAHs and that trans membrane transport is the key determinant for further intracellular biochemical processes [4,11]. Intracellular accumulations were significantly in-
The pH value of the solution might affect both the existing forms and the charge species of PBDEs. Oxygen atom in PBDEs may have a charge of H+ in acid conditions due to the high electronegativity of the atom. Thus, PBDEs in acid solution may have a charge of H+. It seems that the molecular form of PBDEs is easily transported compared with the H+ charged PBDEs. Therefore, we found that the low brominated BDEs had higher transport ratios at pH 7.4 than those at pH 5.5. However, it should be noted that because transport is the result of many factors, such as hydrophobicity, molecular volume, and transporters, this resulted in the high brominated BDEs not showing significantly higher transport ratios at pH 7.4 than those at pH 5.5. For example, organic anion transporting polypeptides (OATPs), highly expressed transporters in mouse liver (also expressed in Caco-2 cells), can transport BDE47, BDE99, and BDE153 [33,34,42]. In addition, human organic anion transporting polypeptide B (OATP2B1) is a pH-sensitive transporter and is expressed in the AP membranes of small intestinal epithelial cells [39]. If these transporters are involved in PBDE transport, more information should be obtained to understand the influence of pH. 3.6. Influence of efflux transporters on transepithelial transport As previously mentioned, the involvement of efflux transporters in the transport of PBDEs cannot be determined based on the influence of concentration alone. To understand the involvement of these transporters, we investigated the roles of three efflux transporters (P-gp, MRP, and BCRP) generally used to study the efflux transport of drugs using the Caco-2 cell monolayer. Three inhibitors, verapamil, MK571 and Ko143, were used to inhibit the expression of the three transporters, respectively, and were added to the chambers. The transport and intracellular accumulation ratios with or without the inhibitors were determined in both directions. The results are shown in Fig. 5 and S3. The transport ratios of PBDEs in the AP-to-BL direction ranged between 0.5 and 15.5% (Fig. 5A), and the intracellular accumulation ratios were between 26.9 and 58.1% when verapamil, a P-gp inhibitor, was added (Fig. 5B). For the BL-to-AP direction, the transport ratios ranged from 0.2 to 4.9%, and the intracellular accumulation ratios 20% P-gp
Transported atios
15%
MRP
BCRP
OCT
A
Control
* ** *
10% *
* *
** **
**
*
5%
*
* * ***
**
**
*
**
** *
*
**
**
** **
**
**
**
* **
0% BDE17
BDE28
BDE71
BDE47
MRP
BCRP
OCT
BDE66
BDE100
BDE99
BDE85
BDE154 BDE153 BDE138 BDE183 BDE190
80% P-gp
B
Control
Accumulated ratios
70% 60% 50% 40% **
30% 20%
**
**
** **
**
** **
**
** **
**
**
10% 0% BDE17
BDE28
BDE71
BDE47
BDE66
BDE100
BDE99
BDE85
BDE154 BDE153 BDE138 BDE183 BDE190
Fig. 5. The effects of efflux and influx transporters on the transepithelial transport and intracellular accumulation of PBDEs (**p < 0.01; *p < 0.05).
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they have low permeability [35]. The present results were consistent with this rule. In the present study, the molecular weights of tri- to penta-BDEs were 406.7–564.5 g/mol, which were lower than those of hexa- (643.4 g/mol) and hepta-BDEs (722.3 g/mol). The present results may indicate that OCTs tend to participate in the transport of tri- to penta-BDEs, while transport of higher brominated congeners is limited. However, further research is required to understand the accumulation of PBDEs in cells after the addition of cimetidine. To further confirm the involvement of the aforementioned transporters during transepithelial transport, further investigations are necessary. For example, the use of gene transfection techniques to establish cell lines with expression of the target genes (the transporters), and subsequent transepithelial transport experiments. 4. Conclusions In the present study, the transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine was determined using a Caco-2 cell monolayer. The results of the present study were showed in Fig. 6. These results suggest the following: (1) passive diffusion dominated the transepithelial transport of PBDEs in the concentration ranges used in this experiment, and transepithelial transport of PBDEs follows pseudo-first-order kinetics; (2) the ratedetermining step of the transepithelial transport of PBDEs is trans-cell transport including the trans-pore process, and the Caco-2 cell monolayer might not be suitable for estimating the bioavailability of chemicals with high LogKOW values; (3) a pH-dependent transport mechanism was involved; (4) the efflux transporters P-gp, MRP, and BCRP might participate in the transepithelial transport of PBDEs; (5) the influx transporters, OCTs, seemed to participate in the transport of tri- to penta-BDEs, while the transport of higher brominated congeners was limited.
Fig. 6. The possible transepithelial transport mechanisms of PBDEs in Caco-2 cell monolayer.
creased in the AP-to-BL direction in the present study. This may be because addition of the inhibitors prevented PBDE efflux from the cells to the AP chamber and caused increased intracellular accumulation. According to the analysis previously mentioned, we assume that PBDEs adsorption onto Caco-2 cells is a rapid process, while the process of entering the cell membrane is slow. The rate-determining step in the transepithelial transport of PBDEs in the Caco-2 cell monolayer is the trans-cell transport, i.e., the transport of PBDEs from the intramembrane of the AP-to-BL side, and vice versa. In addition, considering the very different intracellular accumulation and transepithelial transport ratios between the AP-to-BL and BLto-AP directions, we assumed that the pore of the Transwell insert also had an important effect on the transport ratios of PBDEs. The average membrane pore area in the Transwell insert accounted for less than 0.37% (9 images using three Transwell inserts) of the total area calculated using Nano Measurer software after scanning the surface topography using JSM-7500F (JEOL) scanning electron microscopy (Fig. S4). Thus, we assumed that the rate-determining step of the apparent transepithelial transport ratios may include the trans-pore process (Fig. 6). To estimate the bioavailability of chemicals, both the intracellular accumulation and transepithelial transport of chemicals should be considered [15]. However, we assumed that PBDEs were mainly adsorbed in the phospholipid membrane of the AP side, especially the higher brominated congeners due to high lipophilicity. Actually, the transport of these chemicals was lower. Therefore, we assumed that the Caco-2 cell monolayer might not be suitable for estimating the bioavailability of chemicals with high LogKOW values, such as PBDEs.
Notes The authors declare no competing financial interest. Acknowledgements This research was financially supported by the National Nature Science Foundation of China (Nos. 21277086 and 21677094). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.envres.2016.12.024. References
3.7. Influence of influx transporter on transepithelial transport
Abdallah, M.A., Harrad, S., 2014]. Polybrominated diphenyl ethers in UK human milk: implications for infant exposure and relationship to external exposure. Environ. Int. 63, 130–136. Ahn, S.Y., Eraly, S.A., Tsigelny, I., Nigam, S.K., 2009]. Interaction of organic cations with organic anion transporters. J. Biol. Chem. 284 (45), 31422–31430. Arkoosh, M.R., van Gaest, A.L., Strickland, S.A., Hutchinson, G.P., Krupkin, A.B., Dietrich, J.P., 2015]. Dietary exposure to individual polybrominated diphenyl ether congeners BDE-47 and BDE-99 alters innate immunity and disease susceptibility in juvenile Chinook Salmon. Environ. Sci. Technol. 49 (11), 6974–6981. Bressler, D.C., Gray, M.R., 2003]. Transport and reaction processes in bioremediation of organic contaminants. 1. Review of bacterial degradation and transport. Int. J. Chem. React. Eng. 1, R3. Burkhard, L.P., 2000]. Estimating dissolved organic carbon partition coefficients for nonionic organic chemicals. Environ. Sci. Technol. 34 (22), 4663–4668. Cao, J., Chen, X., Liang, J., Yu, X.Q., Xu, A.L., Chan, E., Duan, W., Huang, M., Wen, J.Y., Yu, X.Y., Li, X.T., Sheu, F.S., Zhou, S.F., 2007]. Role of P-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of Glycyrrhiza glabra. Drug Metab. Dispos. 35 (4), 539–553. Costa, L.G., de Laat, R., Tagliaferri, S., Pellacani, C., 2014]. A mechanistic view of polybrominated diphenyl ether (PBDE) developmental neurotoxicity. Toxicol. Lett. 230 (2), 282–294. Cousein, E., Barthélémy, C., Poullain, S., Simon, N., Lestavel, S., Williame, V., Joiris, E.,
An inhibition assay was carried out in the AP-to-BL direction by adding 50 μM cimetidine, an inhibitor of OCTs (Fig. 5). The transport ratios of tri- to penta-BDEs significantly decreased, which indicated that OCTs may participate in the transport of lower brominated BDEs. In addition, the intracellular accumulation ratios significantly increased compared with the control. OCTs are expressed in the human small intestine. They mediate the transport of some organic compounds including tetraethyl ammonium, acetylcholine, choline, and procainamide [24,48]. In intestinal epithelia, OCTs are present in the lateral membrane (OCT1) and the brush border membrane (OCT3) [13,30,42]. It was reported that OCTs tend to affect the transport of compounds with lower molecular weight, higher hydrophilicity and positively charged compounds [17]. In addition, according to the Rule of Five, chemicals with a molecular weight larger than 500 g/mol are not generally suitable for drugs, as 99
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