Different transport mechanisms for cadmium and mercury in Caco-2 cells: inhibition of Cd uptake by Hg without evidence for reciprocal effects

Cadmium/Hg interactions have been studied in the TC7 clone of the enterocytic-like Caco-2 cells to test the hypothesis that these metals may compete for intestinal transport. Comparison of the kinetic parameter values for 203Hg(II) and 109Cd(II) uptake in a serum-free medium revealed that Hg is accumulated much more rapidly and to higher concentrations. The very rapid uptake/binding step and the initial uptake rate of 109Cd were both significantly inhibited by an excess of unlabeled Cd or Hg (apparent K(i) for Hg of 9.3 +/- 1.2 microM) without reciprocal effects. 109cadmium uptake was highly sensitive to temperature and a significant fraction of accumulation (12%) was EDTA extractable. 203Hg uptake remained insensitive to temperature or the EDTA washing procedure. However, the uptake of both tracers was half-decreased when an excess of the respective unlabeled metal was added in the stop solution, suggesting an exchange mechanism for adsorption. Cell pretreatment with N-ethylmaleimide (NEM) led to a 30% decrease or a 73% increase in the 3-min specific transport of 109Cd when NEM was still present in or removed from the uptake medium, respectively. NEM had no effect on 203Hg uptake. Overall our results suggest the involvement of a saturable specific mechanism for Cd, which is highly sensitive to inhibition by Hg and NEM under some conditions, and a nonspecific passive diffusion for Hg. The Hg- or NEM-induced inhibition of Cd uptake likely involves a thiol-mediated reaction, but our results suggest that NEM pretreatment may activate other cellular mechanisms leading to a stimulatory effect.     

Differential toxicity and gene expression in Caco-2 cells exposed to arsenic species

Inorganic arsenic [As(V) + As(III)] and its metabolites, especially the trivalent forms [monomethylarsonous acid, MMA(III), and dimethylarsinous acid, DMA(III)], are considered the forms of arsenic with the highest degree of toxicity, linked to certain types of cancer and other pathologies. The gastrointestinal mucosa is exposed to these forms of arsenic, but it is not known what toxic effect these species may have on it. The aim of the present work was to evaluate the toxicity and some mechanisms of action of inorganic arsenic and its metabolites [monomethylarsonic acid, MMA(V), dimethylarsinic acid, DMA(V), MMA(III) and DMA(III)] in intestinal epithelial cells, using the Caco-2 human cell line as a model.     

Gastrointestinal absorption of Cd-metallothionein and cadmium chloride in mice

CdCl₂ or Cd-metallothionein (MT) (6 g Cd with 2.25 Ci (83.25 KBq)¹⁰⁹Cd) was given orally to mice, which were sacrificed at 30 min and 2 h after intubation. Although¹⁰⁹Cd in Cd-MT was excreted rapidly into the urine, its absorption was found to be significantly less than that of CdCl₂. The poor absorption was due to a decrease of Cd-MT uptake into the intestine. Cadmium chloride taken up into the mucosa could stimulate MT synthesis even 30 min after its intubation. However, the percentage of MT-bound Cd in the Cd of intestinal supernatants was lower with CdCl₂ (62% at 30 min and 2 h) than with Cd-MT (78% and 84% at 30 min and 2 h, respectively). These results suggest that the transport mode of lumenal Cd-MT to mucosal cells is different from that of lumenal CdCl₂. Lumenal Cd-MT is probably internalized into intestinal cells in an intact form. Furthermore, the Cd-MT may pass through the basolateral membrane in this form. This hypothesis was supported by the different distributions of Cd in the liver and kidney after Cd-MT and CdCl₂ intubations.     

Comparison of anticancer activity between lactoferrin nanoliposome and lactoferrin in Caco-2 cells in vitro

The anticancer activities of Lactoferrin (Lf) and Lf nanoliposomes in Caco-2 cells were observed in this study, and mitochondrial function (MTT assay), count kit-8(CCK-8), detection of intracellular reactive oxygen species (ROS) and apoptosis induction (AO/EB staining) assays were used to evaluate the anticancer activity. MTT results demonstrated that Lf nanoliposomes and Lf reduced the mitochondrial activity of cells in a manner of dose and time effect, and the viabilities of Caco-2 cell were significantly decreased in vitro following exposure to Lf nanoliposomes at the concentrations of 5 and 10 mg/mL. LDH leakage and ROS significantly increased in cells exposed to Lf nanoliposomes (⩾5 mg/mL), while Lf induced ROS only at higher doses (10 mg/mL). CCK-8 evaluation of cell proliferation and AO/EB double staining supported the anti-proliferative effects of Lf liposomes. Our findings demonstrated that the presence of Lf nanoliposome is more significant than Lf in inhibiting human tumor cells proliferation. Therefore, it can be concluded that Lf nanoliposomes are a potential therapeutic modality in the management of tumors.     

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1 > GLUT3 > GLUT5 > GST1A > OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.     

An in vitro approach to assess the toxicity of inhaled tobacco smoke components: nicotine, cadmium, formaldehyde and urethane

One of the first lines of defence to inhaled toxins is the barrier formed by the tracheobronchial epithelium, making this the ideal region for studying the toxicity of inhaled substances. This study utilises a highly differentiated, three-dimensional, in vitro model of human upper respiratory tract epithelium (EpiAirway-100) to measure the acute toxicological responses to well-characterised tobacco smoke components. To determine the suitability of this model for screening inhaled toxicants, the EpiAirway tissue model (ETM) was treated apically with tobacco smoke components (nicotine, formaldehyde, cadmium, urethane) which are known to induce a variety of toxic effects (e.g. cytotoxic, thrombogenic, carcinogenic). A range of concentrations were used to model different mechanisms and severity of toxicity which were then compared to known in vivo responses. Similar trends in stress response occurred, with distinct alterations to the tissue in response to all four toxins. At high concentrations, cell viability decreased and tight junctions were degraded, but at sub-toxic concentrations epithelial resistance (indicating tissue integrity) increased 20-60% from control. This peak in resistance coincided with an increase in secreted protein levels, elevated cytokine release and goblet cell hyperplasia and hypertrophy. In conclusion, acute exposure to tobacco smoke components induces measurable toxic responses within human respiratory epithelium. Sub-toxic concentrations appear to illicit a protective response by increasing mucus secretion and mediating immune responses via cytokine release. These responses are comparable to human in vivo responses, indicating potential for the ETM as a tool for screening the toxicity of inhaled compounds.     

Comparison of the toxicity induced by microcystin-RR and microcystin-YR in differentiated and undifferentiated Caco-2 cells

Cyanobacterial toxins, especially microcystins (MCs), are found in eutrophized waters throughout the world. Acute poisonings on animals and humans have been reported following MC exposure. Around 80 MCs variants have been isolated in surface waters worldwide so far. The toxicity of the most frequent MC congener, MC-LR, is well known; however, studies dealing with MC-RR and MC-YR are less abundant. In this present work, the toxic effects of MC-RR and MC-YR at concentrations of 50, 100, 150 and 200 μM have been investigated in the human colon carcinoma cell line Caco-2 both undifferentiated and differentiated after 24 and 48 h exposure. Toxicity endpoints assessed were cell number by quantification of total protein content of the cell cultures; cell viability by means of neutral red uptake, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) metabolization to detect mitochondrial changes. Moreover, morphological alterations were also investigated. Results showed that protein content was the most sensitive endpoint for MC-RR with reductions of 45% after 48 h exposure to 200 μM MC-RR in differentiated cells (EC₅₀ > 200 μM); whereas for MC-YR is the inhibition of neutral red uptake with reductions higher than 80% at 100 μM in undifferentiated cells after 48 h (EC₅₀ of 57.3 μM). Furthermore, alteration in the cells was shown in the morphological studies, particularly at high concentrations, undergoing general reduction in cell number and hydropic degeneration. The sensitivity of the cultures to these toxins was highly affected by the exposure time and in a lesser extent by the differentiation state, with MC-YR showing higher toxicity than MC-RR.     

Oxidative stress of alternariol in Caco-2 cells

Alternariol (AOH) is a mycotoxin produced by fungus Alternaria. It is found in a wide variety of fruits and cereals products. AOH is able to damage human health. The aim of this study was to evaluate the cytotoxicity of AOH in human colon adenocarcinoma (Caco-2) cells. Moreover, some events related to oxidative stress were evaluated: reactive oxygen species (ROS) generated by oxidation of 2′,7′-dichlorodihydrofluorescein diacetate; peroxidation of lipid (LPO) by malondialdehyde (MDA) production; and antioxidant enzymatic capability of catalase (CAT) and superoxide dismutase (SOD). Cytotoxicity of AOH (from 3.125 to 100 μM) was determined during 24, 48 and 72 h of exposure by different endpoints. AOH decreased cell viability by MTT, NR and PC assays. However, no IC₅₀ values were obtained by any of the assays tested. AOH induced a strong oxidative stress in Caco-2 cells by generation of ROS production and LPO associated with a rise in the SOD activity at all concentration tested. ROS increased 1.2-fold with respect to the control and MDA production ranged from 130% to 250% compared to control. Our results demonstrated that in spite of AOH showing cytotoxic effect on Caco-2 cells at the highest concentration tested, oxidative stress by LPO and ROS was observed at all concentrations assayed. This could cause an injury and be hazardous to health.     

HPLC with programmed wavelength fluorescence detection for the simultaneous determination of marker compounds of integrity and P-gp functionality in the Caco-2 intestinal absorption model

A high sensitivity reversed-phase HPLC method is presented for the simultaneous determination of marker compounds of paracellular transport (atenolol), transcellular transport (propranolol) and P-gp functionality (talinolol) in the Caco-2 system. The Caco-2 system is presently commonly accepted as an in vitro cell culture model of the intestinal mucosa. A programmed wavelength fluorescence detection method was used to optimise the response of the marker compounds. This marker compound mixture and the corresponding HPLC assay can be used for in house validation of the Caco-2 system or to evaluate simultaneously the effect of test compounds or absorption enhancing strategies on monolayer integrity and P-gp functionality. The method can easily be adapted to determine the concentration of atenolol, propranolol and talinolol in blood, thus allowing to use the same compounds in the in situ rat perfusion system with blood sampling from the mesenteric vein.     

Transport of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid and its acyl glucuronide by human intestinal Caco-2 cells

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent cytokine inducer, exhibited marked antitumor activity when given as multiple oral doses in mice. The aim of this study was to examine the transport of DMXAA and its acyl glucuronide (DMXAA-G) using the human Caco-2 cells. DMXAA was minimally metabolized by Caco-2 cells and both DMXAA and DMXAA-G were taken up to a minor extent by the cells. The permeability coefficient (P_app) values of DMXAA over 10–500 μM were 4 × 10⁻⁵ cm/s to 4.3 × 10⁻⁵ cm/s for both apical (AP) to basolateral (BL) and BL-AP transport, while the P_app values for the BL to AP flux of DMXAA-G were significantly greater than those for the AP to BL flux, with R_net values of 4.5–17.6 over 50–200 μM. The BL to AP active efflux of DMXAA-G followed Michaelis-Menten kinetics, with a K_m of 83.5 ± 5.5 μM, and V_max of 0.022 ± 0.001 nmol/min. The flux of DMXAA-G was energy and Na⁺-dependent and MK-571 significantly (P < 0.05) inhibited its BL to AP flux, with an estimated K_i of 130 μM. These data indicate that the transport of DMXAA across Caco-2 monolayers was through a passive process, whereas the transport of DMXAA-G was mediated by MRP1/2.     

金丝桃苷在Caco-2细胞模型中的跨膜转运方式

目的研究金丝桃苷在Caco-2细胞模型中的吸收机制。方法用Caco-2细胞单层模型研究金丝桃苷的双向转运,考察pn、药物质量浓度、方向、温度、抑制剂对金丝桃苷细胞转运的影响。采用HPLC法检测金丝桃苷的含量,计算其表观渗透系数(P_app)。结果金丝桃苷的细胞转运P_app。具有pH依赖性。金丝桃苷肠腔（A）侧→基底（B）侧P_app>B→A,并且随着金丝桃苷质量浓度的增大而减小,具有浓度依赖性。P-gp抑制剂维拉帕米增加金丝桃苷的细胞正向转运P_app,降低了其逆向转运P_app。金丝桃苷较高浓度时,MRPl抑制剂吲哚美辛和ATP抑制剂叠氮化钠显著降低了金丝桃苷的转运量。结论金丝桃苷在Caco-2细胞单层模型中的转运具有pH依赖性和浓度依赖性,是以主动转运为主,被动扩散为辅,同时涉及外排蛋白作用的转运方式。     

Uptake and transport of the ACE-inhibitor ceronapril (SQ 29852) by monolayers of human intestinal absorptive (Caco-2) cells in vitro

The angiotensin-converting enzyme (ACE)-inhibitor ceronapril (SQ 29852) is shown to be a substrate of the intestinal dipeptide transporter. Uptake by Caco-2 cells, grown as confluent monolayers, follows a major saturable pathway (K_m, 0.91 ± 0.11 mM; 90% at 1 mM) together with a minor passive component (k_J, 32.3 ± 6.6 ng (10⁶ cells)⁻¹ (20 min)⁻¹. Uptake was inhibited by competition with dipeptides such as l-AIa-l-Pro (K_i, 2.96 mM) and l-Phe-Gly (K_i, 3.84 mM) but not by cephalosporins such as cephalexin. In contrast, transport was non-saturable, flux increased linearly with concentration and data were consistent with a passive transepithelial transport mechanism. Transport profiles showed a biphasic dependence upon time with an initial flux of 0.83 ± 0.02 ng insert⁻¹ min⁻¹ (k₁) and a terminal value of 1.65 ± 0.08 ng insert⁻¹ min⁻¹ ((k₂) at 100 μM. It is concluded that the basolateral efflux is retarded so that the passive paracellular transport controls the overall transepithelial transport characteristics in the Caco-2 model. Carrier-mediated uptake into intestinal enterocytres, followed by rate-limiting basolateral efflux, may explain the extended t_max in vivo following oral administration.     

Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells

Okadaic acid (OA) is a marine toxin, a tumour promoter and an inducer of apoptosis. It mainly inhibits protein-phosphatases, protein synthesis and enhances lipid peroxidation. Cadmium (Cd) is known to be carcinogenic in animals and humans (group 1 according to the International Agency for Research on Cancer (IARC) classification). Cd also induces oxidative stress in living organisms. Since they are sometimes found simultaneously in mussels, we have evaluated in the present investigation, the lipid peroxidation, as malondialdehyde (MDA) production, in the variation of the ratios of 8-(OH)-dG/10⁵dG and m⁵dC/ (dC + m⁵dC) induced by OA and/or Cd in Caco-2 cells. When cells were treated exclusively by OA (15 ng/ml) or Cd (0.625 and 5μg/ml) for 24 h, protein synthesis was inhibited (by 42 ± 5%, 18 ± 13%, and 90 ± 4% respectively) while MDA production was 2235 ± 129, 1710 ± 20, and 11496 ± 1624 pmol/mg protein respectively. In addition, each toxicant induced modified bases in DNA; increases in oxidised bases and methylated dC. The combination of OA and cadmium was more cytotoxic and caused more DNA base modifications; the ratio m⁵dC/(m⁵dC+dC) was increased from 3 ± 0.15 to 9 ± 0.15 and the ratio 8-(OH)-dG/10⁵ dG also (from 36 ± 2 to 76 ± 6). The combination of OA and Cd also increased the level of MDA (16874 ± 2189 pmole/mg protein). The present results strongly suggest that DNA damage resulting from the oxidative stress induced by these two toxicants may significantly contribute to increasing their carcinogenicity via epigenetic processes. Received: 28 September 1999 / Accepted: 10 January 2000     

神经酰胺对人结肠癌Caco-2细胞中谷胱甘肽硫转移酶A1的影响

谷胱甘肽S-转移酶(GSTs)是一组多功能Ⅱ相药物代谢酶,能够清除外源性的亲电子化合物,包括致癌物、氧化应激产物和化疗药物等。己有研究表明,GSTs的过表达与肿瘤细胞化疗耐药性的产生存在着密切的关系,某些肿瘤耐药细胞株内GSTs含量要比敏感株高出许多倍,肿瘤细胞可通过表达GSTs而保护自身免受化疗药物的损害,是机体防御机制的     

Estimation of hydroxymethylfurfural availability in breakfast cereals. Studies in Caco-2 cells.

The transport and availability of hydroxymethylfurfural (HMF), an intermediary product of the Maillard reaction, was investigated in the Caco-2 cell line after in vitro gastrointestinal digestion. The study was carried out at two levels; (a) an HMF-spiked culture medium, and (b) digested commercial breakfast cereals (BC). In both assays, the higher the amount of HMF offered to the cells, the higher the absolute value of transported HMF. However, HMF availability and transport are not directly proportional to the initial HMF content since HMF is partly retained in the non-soluble fraction after digestion. In addition, HMF is degraded to some extent during the gastrointestinal digestion of both HMF-spiked cell medium and BC. Average HMF availability from three commercial breakfast cereals was 9.1% (4.98-12.99%). Variations in HMF availability may be related to the particular composition of each BC, where fibre could play an important role. On the other hand, possible metabolization into the cell should also be considered.     

Protective effect of sodium molybdate against the acute toxicity of cadmium chloride

Pretreatment of rats with Na₂MoO₄ (1.24 mmol/kg, once a day for 3 days, i.p.) partially protected them againts the acute toxicity of CdCl₂ (0.075 mmol/kg, once, s.c., 24 h after pretreatment with Na₂MoO₄). The survival number of rats per total number of rats in the CdCl₂-dosed group was 10/10, 8/10, 6/10, 2/10 and 0/10 on 0, 1, 2, 6 and 18 days after treatment with CdCl₂ whereas in the group where CdCl₂ is given after pretreatment with Na₂MoO₄ it is 10/10 and 6/10 on 0 and 18 days. The body weight of CdCl₂-dosed rats consistently decreased until their death while that of Na₂MoO₄- CdCl₂-dosed rats similarly decreased up to 4 days after exposure to CdCl₂ but then increased almost normally. In order to elucidate the mechanism of protective action of Na₂MoO₄ against the acute toxicity of CdCl₂, cellular components such as DNA, inorganic cations and metallothionein were measured in the liver after exposure to CdCl₂. The treatment with CdCl₂ alone reduced K content and increased Ca content but pretreatment with Na₂MoO₄ prevented such alterations in the levels of those cations caused by CdCl₂. Metallothionein content in the liver was significantly elevated in the CdCl₂-treated groups as compared to saline controls although the protein content was higher in the Na₂MoO₄-CdCl₂-dosed group than in the CdCl₂-dosed group. There was no difference in the protein content of the liver between saline controls and the Na₂MoO₄-dosed group. This suggests that Na₂MoO₄ alleviated the acute toxicity of CdCl₂ in the rat and the protective mechanism by the metal is in part related to the enhancement of liver Cd-metallothionen induction.     

Ochratoxin A secretion by ATP-dependent membrane transporters in Caco-2 cells

The ATP-dependent membrane transporters, P-gp, MRP2 and BCRP, localized in the luminal membranes of the intestines, liver and kidney, counteract absorption and increase excretion of xenobiotics and drugs. Previously, it has been suggested that the mycotoxin ochratoxin A (OTA) is a substrate for ATP-dependent transporters, and hence the absorption and secretion of OTA in the Caco-2 cell model was investigated. To this end, Caco-2 cells were cultured as confluent monolayers in bicameral inserts and the transepithelial transport of the mycotoxin was assessed. Caco-2 cells secreted OTA to the luminal side in a concentration-dependent manner. This secretory permeability was higher than the absorptive permeability, while the absorptive permeability remained constant for all OTA concentrations tested. The secretion decreased and absorption increased in the presence of the MRP-inhibitor MK571, the P-gp and BCRP inhibitor GF120918, and the BCRP-inhibitor Ko143, suggesting that the secretion of OTA is mediated by MRP2 and BCRP. Cyclosporine A also decreased the secretory permeability, but did not affect absorptive permeability, while PSC833 did neither change absorption nor secretion of OTA. Hence it can be suggested that OTA is a substrate for MRP2 as well as BCRP. These findings are of interest in evaluating mycotoxin absorption after oral ingestion, tissue distribution and particularly excretion pathways, including renal, biliary and mammary gland excretion.