Mol. Hum. Reprod. Advance Access originally published online on November 8, 2006
Molecular Human Reproduction 2007 13(1):77-83; doi:10.1093/molehr/gal092
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Expression and characterization of vitamin C transporter in the human trophoblast cell line HTR-8/SVneo: effect of steroids, flavonoids and NSAIDs
1Department of Biology, Section of General Physiology, 2Department of Pharmaceutical Sciences and 3Department of Biomedical Sciences and Advanced Therapy, Section of Obstetrics and Gynaecology, University of Ferrara, Ferrara, Italy
4 To whom the correspondence should be addressed at: via L.Borsari, 46, 44100 Ferrara, Italy. E-mail: clm{at}unife.it
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Abstract |
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Vitamin C plays an important role in embryogenesis and fetal growth as well as in the progression of pregnancy and delivery. Therefore, it is important to understand the mechanism that mediates its transport to the fetus as well as the possible influences by endogenous and exogenous substances on its placental uptake. The aim of this study was to investigate placental sodium-dependent vitamin C transporters (SVCT) 1 and 2. By means of RT–PCR, we found that SVCT2, but not SVCT1, mRNA is expressed in human trophoblast cell line HTR-8/SVneo. Our method was able to confirm SVCT2 mRNA expression in human first-trimester chorionic villi but not in term placental tissue. Cell line kinetic studies of [14C] ascorbic acid (AA) uptake indicated a one-site model and a saturable process. Fetal bovine serum (FBS) and epidermal growth factor (EGF) do not influence the transport properties, although they significantly increase the expression of SVCT2. Steroid hormones (17ß-estradiol, progesterone and cortisol), flavonoids (genistein and quercetin) and non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin and diclofenac) inhibit [14C]AA uptake in a dose-dependent and non-competitive manner. On the contrary, the process is not influenced by aspirin. Our study suggests the use of HTR-8/SVneo cells as a suitable model for trophoblast vitamin C transport investigation.
Key words: ascorbate/NSAIDs/flavonoids/steroids/trophoblasts
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Introduction |
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Vitamin C, when present in its reduced form, ascorbic acid (AA), by acting as an anti-oxidant and as a co-factor in the synthesis of extracellular matrix proteins, regulates trophoblastic function, embryogenesis, fetal growth and the progression of pregnancy and delivery (Prasad et al., 1998
; Jauniaux et al., 2004; Biondi et al., 2005). The first-trimester human placenta has limited antioxidant enzyme capacity and therefore, at this time, the embryo is most vulnerable to oxidative stress. The fetus receives antioxidant molecules, among which vitamin C, from the uterine glands and the secondary yolk sac (Jauniaux et al., 2004). The indispensable need for AA during prenatal life has been shown in mice deficient in the AA transporter Slc23a1 (Sotiriou et al., 2002), orthologue of the rat AA transporter sodium-dependent vitamin C transporters (SVCT2) (Tsukaguchi et al., 1999). Indeed, these newborn mice, Slc23a1–/–, die of respiratory failure and intra-parenchymal brain haemorrhage within a few minutes of birth.
As yet, it is known that L-AA transport is mediated by two different sodium-dependent proteins, SVCT1, mainly expressed in the intestine, liver and kidney epithelia, and SVCT2 expressed in other tissues (Tsukaguchi et al., 1999). At the placental level, SVCT2-mediated transport seems to be operative, and SVCT2 cDNA has been isolated from a human placental choriocarcinoma cell line library, whose expression in human retinal pigment epithelial (HRPE) cells is able to induce Na+ gradient-dependent ascorbate uptake (Rajan et al., 1999). This induced transport activity exhibits similar features to those previously described in JAR human placental choriocarcinoma cell line (Prasad et al., 1998), and it has been suggested that such an active transport mechanism could account for the much higher concentration of AA in the fetus, as compared with maternal blood (Rice, 2000).
Taking into account the supposed role of vitamin C in feto-maternal homeostasis throughout pregnancy, as well as the influence exerted by several substances on its transport under various experimental conditions, we decided to investigate the features of trophoblastic AA uptake. In this study, we tested the suitability of the human trophoblast cell line HTR-8/SVneo as a model for the in vitro study of vitamin C placental transport. For this purpose, we searched for SVCT1 and SVCT2 mRNA in the above-mentioned cell line, as well as in human first-trimester chorionic villi and placental tissue at term. Furthermore, we investigated the effect of the following substances on SVCT2 expression and activity in the trophoblast cell line: fetal bovine serum (FBS), epidermal growth factor (EGF), 17ß-estradiol, progesterone and cortisol. As regards to these steroid hormones, that are involved in the control of pregnancy, their eventual effect on vitamin C placental transport, to our knowledge, has never been investigated. We also tested competition for vitamin uptake by genistein and quercetin. These flavonoids, which are commonly ingested by the diet, are reported to influence SVCT1- and SVCT2-mediated AA transport in different cellular models (Kuo et al., 1997; Park and Levine, 2000; Song et al., 2002). Finally, we tested the effect of aspirin, indomethacin and diclofenac, anti-inflammatory drugs frequently administered for therapeutic purposes during pregnancy. Indeed, some of these compounds have already been shown to influence SVCT2-mediated AA transport in other cells (Manfredini et al., 2002; Dalpiaz et al., 2004).
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Materials and methods |
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[14C]AA (specific activity 6 mCi/mmol) was obtained from NEN Life Science, Boston, MA, USA. Tissue culture media, sera, RNA extraction and RT–PCR reagents were purchased from Invitrogen (Paisley, Scotland, UK).
Steroids, flavonoids, NSAIDs and dithiothreitol (DTT) were from Sigma, St. Louis, MO, USA. qPCR human reference cDNA, random-primed, was purchased from Clontech Laboratories, Mountain View, CA, USA.
All other chemicals were the highest reagent grades commercially available.
HTR-8/SVneo cell culture and treatment
The HTR-8/SVneo trophoblast cell line was kindly provided by Dr CH Graham, Queen’s University, Kingston, Ontario, Canada. Cells were cultured at 37°C in an atmosphere of 5% CO2/95% air in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% FBS and 2% penicillin/streptomycin. For SVCT2 mRNA expression and SVCT2 interaction studies, cells were seeded in 6-multiwell and 24-multiwell plates, respectively, grown to 100% cell density at confluence and pre-exposed 24 h before the experiments to RPMI 1640 and supplemented with 10% FBS, or serum-free, or serum-free plus 25 ng/ml EGF.
Tissue preparation
For RNA extraction studies, first-trimester human chorionic villi were obtained from four patients undergoing chorionic villous biopsy for prenatal diagnosis at the 11th week of gestation. In these cases, the small amount necessary for the assay derived from the tissue discarded by the geneticist. Term placental tissue was obtained from physiological pregnancies with appropriately grown singleton fetuses, by elective caesarean section in patients, which had been delivered by caesarean section in the previous pregnancy. All the patients gave verbal informed consent to the study. Approval for the experimental protocol was obtained from the University of Ferrara institutional review board. Tissue samples were immediately frozen in liquid nitrogen and stored at –80°C.
RT–PCR
Total RNA was isolated from HTR-8/SVneo cells, human chorionic villi and placental tissue at term using Trizol reagent (Invitrogen) as manufacturer’s suggested procedure. RT–PCR was employed to search for the expression of SVCT1 and SVCT2 transporter isoforms. First-strand cDNA was synthesized by incubating 5 µg RNA with 0.5 µg oligo(dt)12–18, at 70°C for 10 min and subsequently a reaction mix of avian myeloblastosis virus reverse-transcriptase (AMV RT) buffer, 500 mM of dNTPs, 2.5 units of AMV RT was added. The samples were then incubated at 45°C for 50 min. cDNA was kept at –20°C until used for PCR.
The synthesized cDNA was amplified in a standard PCR reaction mix containing 200 µM dNTP, 1.5 mM MgCl2, 0.25 µg of each sense and antisense primer and 0.25 units of Taq DNA polymerase. Primer sequences were examined for specificity by comparison with known gene sequences in GenBank using the blastn program at the BLAST website (http://www.ncbi.nlm.nih.gov/BLAST/). PCR amplification from reverse transcribed cDNA was carried out using specific PCR primers for SVCT1 and SVCT2. SVCT1 sense: 5'-GCCCCTGAACACCTCTCATA-3'; SVCT1 antisense: 5'-ATGGCCAGCATGATAGGAAA-3'; SVCT2 sense: 5'-TTCTGTGTGGGAATCACTAC-3'; SVCT2 antisense: 5'-ACCAGAGAGGCCAATTAGGG-3' (Garcia et al., 2005). qPCR human reference cDNA (Clontech) was used as positive control template for validation of gene primer design.
PCR amplification for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA was performed on the same samples as a parallel control. GAPDH sense: 5'-CCACCCATGGCAAATTCCATGGCA-3'; GAPDH antisense: 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (Xue et al., 1996). A 1/20 volume of the generated cDNA reaction was used in the amplification reaction. PCR was performed in a 25 µl volume using 1.5 mM magnesium chloride, 0.2 mM dNTPs, 0.25 mg of each sense and antisense primer and 0.25 units of Taq polymerase. PCR for both SVCT1 and SVCT2 was performed for 20–40 cycles of amplification using the following parameters: denaturation at 94°C for 20 s, annealing at 55°C for 30 s and extension at 72°C for 40 s. The curve for SVCT1 and SVCT2 amplificates showed linear portions for 26–30 cycles, and 28 cycles were used in the semi-quantitative analysis. PCR amplification for GAPDH was performed on the same samples as a parallel control, with a linear range of amplification between 20 and 26 cycles, and 23 cycles were used in the semi-quantitative analysis under the following PCR conditions: denaturation at 90°C for 20 s, annealing at 55°C for 30 s and extension at 72°C for 40 s. The amplified products, of length 339 bp for SVCT2 and 598 bp for GAPDH, were separated on a 1.5% agarose gel containing ethidium bromide, using a DNA ladder as size marker.
After gel electrophoresis, scanning densitometry of the areas of interest was performed using the ‘Gel Doc 2000’ video imaging system (Bio-Rad Laboratories, Hercules, CA, USA) to obtain the arbitrary density units. Band densities corresponding to SVCT2 expression following several treatments were quantified using Quantity One software (Bio-Rad Laboratories), and the ratio of SVCT2 to GAPDH band intensities was used as an indication of the relative abundance of the two templates in the samples.
[14C]Ascorbate uptake and SVCT2 transporter interactions
Transport assays were performed following the method described by Rajan et al. (1999). In brief, the uptake buffer was prepared fresh each time, and the composition was 25 mM Hepes/Tris (pH 7.5), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4 and 5mM glucose. DTT (1 mM) was also added to the uptake buffer to prevent the oxidation of AA. At this concentration, DTT had no effect on the transport process. The incubation time for the transport measurements was 60 min, within the time course of linear uptake (data not shown), at 37°C, after this time the uptake buffer containing the radioactive substrate was aspirated off, and cells were washed with 2 x 2 ml of ice-cold uptake buffer. Cells were then dissolved in 250 µl of 0.2 M NaOH solution containing 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1 propanesulfonate (CHAPS) (Sigma), transferred to vials, and radioactivity associated with the cells was evaluated by liquid scintillation spectrometry.
The kinetics of SVCT2-mediated [14C]AA uptake were analysed, performing a homologous competition experiment. It consisted of a concentration-response curve where unlabeled AA ranged from 2.5 to 1000 µM, and the concentration of [14C]AA ranged from 2.5 to 50 µM, being kept constant at 50 µM. Data were analysed by nonlinear regression of the Michaelis–Menten equation and confirmed by Eadie–Hofstee linear regression.
Inhibition of AA transport was determined by adding the indicated concentrations of unlabelled compounds to plated cells along with either [14C]AA at a fixed concentration of 50 µM or [14C]AA ranging from 2.5 to 100 µM. The unlabelled inhibitor concentrations displacing 50% of [14C]AA (IC50 values) were obtained by computer analysis of displacement curves. Inhibitory binding constants (Ki values) were derived from the IC50 values according to the Cheng and Prusoff equation Ki = IC50/(1+[C*]/ Kt*), where [C*] is the concentration of [14C]AA and Kt* its Michaelis–Menten constant (Cheng and Prusoff, 1973). Competition studies were performed according to Lineweaver–Burk analysis. All calculations were performed using the Graph Pad Prism (GraphPad) computer programme.
Data analysis
Statistical analysis was performed by ANOVA followed by Dunnett’s t-test. The difference was considered statistically significant at P < 0.05.
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Transporter expression
We analysed the expression of AA transporter mRNA in HTR-8/SVneo cells, human chorionic villi and placental tissue at term by RT–PCR, using the set primers previously reported by Garcia et al. (2005)
for both SVCT1 and SVCT2. We found that the amplified DNA band corresponding to 339 bp was the expected size for the amplification product of SVCT2, whereas no amplification product was detected with the specific primers for SVCT1, corresponding to 360 bp. This result was obtained for HTR-8/SVneo cells and chorionic villi, whereas no PCR products were detected in placental tissue at term (Figure 1A). The commercially available human reference cDNA, the positive control, showed two bands of 339 and 360 bp as amplification products of SVCT2 and SVCT1, under each PCR conditions, respectively (Figure 1B).
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[14C]AA uptake studies
The rate of [14C]AA uptake by HTR-8/SVneo cells is hyperbolically related to its concentration, thus indicating a saturable transport process. Furthermore, the Eadie–Hofstee linear regression of data was linear in the range of concentration used (r = 0.997; P < 0.0001), and analysis of the saturation curve indicates a one-site rather than two-site model. It appears therefore that these cells express a single class of transporter, with a Michaelis–Menten constant (Kt*)= 45.0 ± 3.0 µM and a Vmax = 2.8 ± 0.2 nmoles/106 cells/60 min (Figure 2).
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Inhibition of SVCT2-mediated uptake
We studied the influence of AA, 17ß-estradiol, progesterone, cortisol, genistein, quercetin, aspirin, indomethacin and diclofenac on [14C]AA uptake (50 µM) by HTR-8/SVneo cells. In Figure 3A (left), the inhibition curves of [14C]AA uptake in the presence of increasing concentrations of AA, 17ß-estradiol, progesterone and cortisol are reported. Calculation of the inhibition constants (Ki) demonstrates that, among steroids, the highest affinity for SVCT2 is expressed by 17ß-estradiol (Ki = 6.2 ± 0.3 µM) and the lowest by progesterone (Ki = 64.0 ± 5.0 µM). The Ki for endogenous substrate AA was 23.0 ± 3.0 µM. On the right of the Figure 3A, the Lineweaver–Burk plots show that the most potent inhibitor, 17ß-estradiol, inhibited SVCT2-mediated uptake in a non-competitive manner.
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The inhibition curves of [14C]AA uptake obtained in the presence of the flavonoids genistein and quercetin are reported in Figure 3B (left). The highest affinity (Ki = 7.4 ± 0.8 µM) was shown by quercetin, with which a non-competitive inhibition was exhibited (Figure 3B, right).
As for the NSAID–SVCT2 interaction, both indomethacin and diclofenac were shown to inhibit [14C]AA uptake (Figure 3C, left). The highest affinity for the SVCT2 transporter was shown by diclofenac (Ki = 5.3 ± 0.6 µM) which behaved as a non-competitive inhibitor (Figure 3C, right). Furthermore, Figure 3C, on the left, highlights that aspirin did not interact with SVCT2.
The highest concentration of DMSO and ethanol (0.1% v/v) had no effect on [14C]AA uptake.
Modulation of SVCT2 mRNA expression
Twenty-four-hour cell culture in a medium containing FBS (10% v/v) induced a significant increase (2.3-fold; P < 0.001) of SVCT2 mRNA expression with respect to the serum-free condition (Figure 4). Treatments with EGF and FBS of cultures at complete confluence did not alter cell number compared with serum-free control cells. Cell viability remained at 98–100% in all experiments. Cell number, total cellular protein and total cellular RNA per well were also unaffected (data not shown). Taking into account the ubiquitous effect of serum on gene expression, the housekeeping GAPDH gene was used for normalization of target gene values throughout experiments, because its level did not vary significantly as a function of time or serum and EGF stimulation; indeed, ratios of SVCT2 mRNA to GAPDH mRNA remain virtually constant. A comparable increase (2.2-fold; P < 0.001) was elicited by adding EGF (10 ng/ml) to the serum-free medium (Figure 4). Twenty-four-hour cell treatment with 17ß-estradiol, progesterone and cortisol at 10–6M did not modify SVCT2 mRNA expression (data not shown).
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Modulation of SVCT2-mediated uptake
Pre-treatment of HTR-8/SVneo cells for 24 h with FBS (10% v/v), as well as with EGF (10 ng/ml), increased the Vmax, although to different extents (Figure 5A), without affecting the Kt value (Figure 5B). Figure 6 shows the statistical analysis of Vmax and Kt values obtained in the presence and absence of serum and EGF. In the presence of FBS, Vmax significantly increases (200%; P < 0.001) with respect to the serum-free condition; similarly, EGF significantly increases Vmax (166%; P < 0.05) (Figure 6A). FBS and EGF treatment did not affect Kt values (Figure 6B). Pre-treatment with 10–6 M steroid hormones for 24 h did not change the SVCT2-mediated transport parameters (Figure 7).
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Discussion |
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It is well known that oxidative processes play a fundamental role in pregnancy because of their influence upon the vascular changes in the maternal organism, as well as on the regulation of uterine and cervical tone during gestation and delivery (Biondi et al., 2005
). Vitamin C represents a classical anti-oxidant component of the oxidative balance, exerting its regulatory influence throughout the entire course of gestation (Prasad et al., 1998; Jauniaux et al., 2004; Biondi et al., 2005), although its physiological role is not yet completely understood. Several early and late complications of pregnancy, from fetal malformation to pre-eclampsia, can be interpreted as a consequence of hypoperfusion of the product of conception (Vesce et al., 1997) and are related to the inflammatory changes following derangement of the oxidative balance (Biondi et al., 2005). Therefore, it is possible that AA exerts a protection against these conditions by reducing utero-placental vascular impairment induced by oxidative stress, thus preventing inflammation. Several experimental and clinical evidences enforce the concept of a protective role of vitamin C during pregnancy. AA influences trophoblastic apoptosis (Kamudhamas et al., 2004), and it appears to prevent diabetic (El-Bassiouni et al., 2005) as well as ethanol-induced embryopathy (Peng et al., 2005). Furthermore, respiratory failure and intra-parenchymal brain haemorrhage observed in newborn mice Slc23a1–/– (Sotiriou et al., 2002) represent features of human premature birth as well, the risk of which is significantly elevated in a variant in the SVCT SLC23A2 (Erichsen et al., 2006). Moreover, it is known that the placenta is a source of free radical synthesis (Raijmakers et al., 2004), which plays an important role in the pathogenesis of pre-eclampsia (Redman and Sargent, 2005), and it is suggested that vitamin C may exert a protective role in this condition (Chappell et al., 1999; Raijmakers et al., 2004). However, there is controversy about the possible efficacy of vitamin C administration in pre-eclampsia (Poston et al., 2006), probably because of the difficulty in setting trials free from clinical bias.
Our attempts to demonstrate the expression of placental SVCT2 mRNA throughout pregnancy were only in part successful. Indeed, we found that in the human trophoblast cell line HTR-8/SVneo and in human first-trimester chorionic villi, the AA transporter SVCT2, but not SVCT1, mRNA is expressed. Such a result is consistent with the isolation of SVCT2 cDNA from a human placental choriocarcinoma cell line library (Rajan et al., 1999), which represents by definition early pregnancy cancer tissue. As for advanced pregnancy, we could not obtain normal human second-trimester placental tissue. Furthermore, as regards normal placental tissue at term, although we strictly observed the technical rules aimed at avoiding RNA degradation (Haimov-Kochman et al., 2006), in our hands RT–PCR technique was unable to show the expression of the transporter mRNA. At the moment, we do not have any explanation for such a result. Because SVCT2 specific transcript was found by northern blot analysis in commercially available placental tissue of unknown gestational age by Rajan et al. (1999), the transporter mRNA expression in term placental tissue deserves further investigation. To our knowledge, the transporter has never been studied in primary cell cultures. However, due to our failure to demonstrate both SVCT1 and SVCT2 mRNA expression in term placental tissue, at the moment the interpretation of our results must be limited to early pregnancy.
The kinetics of [14C]AA uptake by HTR-8/SVneo cells show properties similar to SVCT2-mediated transport described in other human cells (Prasad et al., 1998; Rajan et al., 1999; Manfredini et al., 2002). Our study also shows that the expression of the AA transporter mRNA is under the control of growth factors, as revealed by its up-regulation by serum, as well as by EGF, addition to the culture medium. Accordingly, pre-treatment with FBS and EGF induces a significant increase in AA uptake Vmax without changing the affinity for the substrate, thereby suggesting an increase in transporter number.
Besides transporter identification, we were also interested in investigating the possibility of influencing its activity. To this end, we choose three classes of substances, namely steroid hormones involved in the control of pregnancy, flavonoids commonly ingested in the diet and NSAIDs, which are frequently used for therapeutic purposes. We found that all tested compounds, except for aspirin, inhibited [14C]AA uptake by HTR-8/SVneo cells in a non-competitive manner, namely binding to the transporter in a different site from that of endogenous ligand ascorbate, whose velocity of transport (Vmax) was therefore reduced. Among the steroids, 17ß-estradiol exhibited the strongest inhibitory action, as did quercetin among the flavonoids and diclofenac among the NSAIDs. All tested substances, except for progesterone, show a Ki value lower than that of the endogenous substrate AA. As for progesterone, the tissue concentration of the hormone can reach up to 5000 ng/gm in the placenta throughout pregnancy (Siiteri and Stites, 1982). This concentration, which corresponds to 
10–20 µM, i.e. lower than progesterone Ki, should not be able to significantly inhibit AA uptake by SVCT2. However, it is worth outlining that in some gestational complications such as threatened or recurrent abortion, progesterone can be prescribed at doses as high as 800 mg daily for several weeks. In this condition, the hormone could possibly reach the tissue concentration able to inhibit AA uptake. The same consideration should be made for 17ß-estradiol and for cortisol, which show a Ki about 10 and 6 times lower, respectively, when compared with progesterone.
As regards to flavonoids, our results are consistent with the inhibitory action by different compounds on the human intestinal ascorbate transporter SVCT1 observed in transfected chinese hamster ovary (CHO) cells and Xenopus laevis oocytes (Song et al., 2002) and in some intestinal cell lines (Kuo et al., 1997), as well as in other SVCT2-expressing human cell lines (Park and Levine, 2000). Indeed, we found that quercetin and genistein inhibited SVCT2-mediated ascorbate uptake by HTR-8/SVneo cells in a non-competitive manner. However, the concentration required for inhibitory action is much higher (Ki = 7.4 µM for quercetin and Ki = 28.0 µM for genistein) than that reached systemically by dietary ingestion. Instead, such a concentration is realistic in the intestine, and therefore, flavonoids may impair maternal AA absorption at this level, thus decreasing trophoblastic availability of the vitamin (Song et al., 2002).
Finally, it is important to note the behaviour of plasma therapeutic concentrations (Giagoudakis and Markantonis, 2005) of the NSAIDs in the light of their possible use for clinical purposes during pregnancy. As for diclofenac, our data show a non-competitive inhibitory action on [14C]AA uptake by HTR-8/SVneo cells similar to that exhibited in HRPE cells (Manfredini et al., 2002; Dalpiaz et al., 2004), with a Ki about four times lower than that of indomethacin. Most interestingly, aspirin, which has been reported to slightly inhibit [14C]AA uptake in oocytes expressing SVCT-1 or SVCT-2 (Tsukaguchi et al., 1999), did not exert any influence on its uptake by HTR-8/SVneo cells. Although both diclofenac and indomethacin, along with other cyclo-oxygenase inhibitors, have been reported to induce several adverse effects on fetal development and well-being (Chan et al., 2001; Auer et al., 2004; Benini et al., 2004; Friedman et al., 2005), they are still in use as tocolytic agents (Vermillion and Robinson, 2005; Loe et al., 2005). In our opinion, the inhibitory action of diclofenac and indomethacin on SVCT2-mediated transport of AA should be added to their adverse effects, suggesting caution in their use during pregnancy. On the contrary, aspirin, which is widely prescribed for the prevention of several complications of pregnancy (Rumbold et al. 2005), appears to be the drug of choice as regards AA trophoblastic uptake in our data. Taken together, our results should suggest to preserve AA levels in the fetal compartment through maternal supplementation of the vitamin, carefully considering the impairment of its uptake by dietary components, as well as by compounds administered for therapeutic purposes.
The results of this study need to be confirmed using trophoblast primary cell cultures as well as models for in vivo investigation. Nevertheless, our experiments show that HTR-8/SVneo cells represent a suitable model for the first trimester in vitro study of AA transport and SVCT2 protein.
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Acknowledgements |
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The authors thank Anna Forster for the English revision of the text. This work was supported with grants from the following Institutions: the University of Ferrara; the Public Health Ministry and the Education and University Ministry of the Republic of San Marino; ‘Telefonia Mobile Sammarinese’ and the ‘Fondazione Cassa di Risparmio’ di Cento, Italy.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Rumbold A, Middleton P and Crowther CA. (2005) Vitamin supplementation for preventing miscarriage. Cochrane Database Syst Rev 2,CD004073.
Auer M, Brezinka C, Eller P, Luze K, Schweigmann U and Schwarzler P (2004) Prenatal diagnosis of intrauterine premature closure of the ductus arteriosus following maternal diclofenac application. Ultrasound Obstet Gynecol 23,513–516.[CrossRef][Web of Science][Medline]
Benini D, Fanos V, Cuzzolin L and Tato L (2004) In utero exposure to nonsteroidal anti-inflammatory drugs: neonatal renal failure. Pediatr Nephrol 19,232–234.[CrossRef][Web of Science][Medline]
Biondi C, Pavan B, Lunghi L, Fiorini S and Vesce F (2005) The role and modulation of the oxidative balance in pregnancy. Curr Pharm Des 11,2075–2089.[CrossRef][Web of Science][Medline]
Chan LY, Chiu PY, Siu SS and Lau TK (2001) A study of diclofenac-induced teratogenecity during organogenesis using a whole rat embryo culture model. Hum Reprod 16,2390–2393.
Chappell LC, Seed PT, Briley AL, Kelly FJ, Lee R, Hunt BJ, Parmar K, Bewley SJ, Shennan AH, Steer PJ et al. (1999) Effect of antioxidants on the occurrence of pre-eclampsia in women at increased risk: a randomised trial. Lancet 354,810–816.[Web of Science][Medline]
Cheng YC and Prusoff WH (1973) Relationships between the inhibition constant (ki) and the concentration of inhibitor which cause 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22,3099–3108.[CrossRef][Web of Science][Medline]
Dalpiaz A, Pavan B, Scaglianti M, Vitali F, Bortolotti F, Biondi C, Scatturin A, Tanganelli S, Ferraro L, Prasad P et al. (2004) Transporter-mediated effects of diclofenamic acid and its ascorbyl pro-drug in the in vivo neurotropic activity of ascorbyl nipecotic acid conjugate. J Pharm Sci 93,78–85.[CrossRef][Web of Science][Medline]
El-Bassiouni EA, Helmy MH, Abou Rawash N, El-Zoghby SM, Kamel MA and Abou Rayah AN (2005) Embryopathy in experimental diabetic gestation: assessment of oxidative stress and antioxidant defence. Br J Biomed Sci 62,71–76.[Web of Science][Medline]
Erichsen HC, Engel SA, Eck PK, Welch R, Yeager M, Levine M, Siega-Riz AM, Olshan AF and Chanock SJ (2006) Genetic variation in the sodium-dependent vitamin C transporters, SLC23A1, and SLC23A2 and risk for preterm delivery. Am J Epidemiol 163,245–254.
Friedman S, Flidel-Rimon O, Steinberg M and Shinwell ES (2005) Indomethacin tocolysis and white matter injury in preterm infants. J Matern Fetal Neonatal Med 18,87–91.[CrossRef][Web of Science][Medline]
Garcia MA, Salazar K, Millàn C, Rodriguez F, Montecinos H, Caprile T, Silva C, Cortes C, Reinicke K, Vera JC et al. (2005) Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells. Glia 50,32–47.[CrossRef][Web of Science][Medline]
Giagoudakis G and Markantonis SL (2005) Relationships between the concentrations of prostaglandins and the nonsteroidal anti-inflammatory drugs indomethacin, diclofenac, and ibuprofen. Pharmacotherapy 25,18–25.[CrossRef][Web of Science][Medline]
Haimov-Kochman R, Fisher SJ and Winn VD (2006) Modification of the standard Trizol-based technique improves the integrity of RNA isolated from RNase-rich placental tissue. Clin Chem 52,159–160.
Jauniaux E, Cindrova-Davies T, Johns J, Dunster C, Hempstock J, Kelly FJ and Burton GJ (2004) Distribution and transfer pathways of antioxidant molecules inside the first trimester human gestational sac. J Clin Endocrinol Metab 89,1452–1458.
Kamudhamas A, Pang L, Smith SD, Sadovsky Y and Nelson DM (2004) Homocysteine thiolactone induces apoptosis in cultured human trophoblasts: a mechanism for homocysteine-mediated placental dysfunction? Am J Obstet Gynecol 191,563–571.[CrossRef][Medline]
Kuo SM, Morehouse HF Jr and Lin CP (1997) Effect of antiproliferative flavonoids on ascorbic acid accumulation in human colon adenocarcinoma cells. Cancer Lett 116,131–137.[CrossRef][Web of Science][Medline]
Loe SM, Sanchez-Ramos L and Kaunitz AM (2005) Assessing the neonatal safety of indomethacin tocolysis: a systematic review with meta-analysis. Obstet Gynecol 106,173–179.[Web of Science][Medline]
Manfredini S, Pavan B, Vertuani S, Scaglianti M, Compagnone D, Biondi C, Scatturin A, Tanganelli S, Ferraro L, Prasad P et al. (2002) Design, synthesis and activity of ascorbic acid prodrugs of nipecotic, kynurenic and diclophenamic acids, liable to increase neurotropic activity. J Med Chem 45,559–562.[CrossRef][Web of Science][Medline]
Park JB and Levine M (2000) Intracellular accumulation of ascorbic acid is inhibited by flavonoids via blocking of dehydroascorbic acid and ascorbic acid uptakes in HL-60, U937 and jukat cells. J Nutr 130,1297–1302.
Peng Y, Kwok KH, Yang PH, Ng SS, Liu J, Wong OG, He ML, Kung HF and Lin MC (2005) Ascorbic acid inhibits ROS production, NF-kappa B activation and prevents ethanol-induced growth retardation and microencephaly. Neuropharmacology 48,426–434.[CrossRef][Web of Science][Medline]
Poston L, Briley AL, Seed PT, Shennan AH for the Vitamins in Pre-eclampsia (VIP) Trial Consortium (2006) Vitamin C and E in pregnant women at risk for pre-eclampsia (VIP trial): randomized placebo-controlled trial. Lancet 367,1145–1154.[CrossRef][Web of Science][Medline]
Prasad PD, Huang W, Wang H, Leibach FH and Ganapathy V (1998) Transport mechanisms for vitamin C in the JAR human placental choriocarcinoma cell line. Biochim Biophys Acta 1369,141–151.[Medline]
Raijmakers MT, Dechend R and Poston L (2004) Oxidative stress and pre-eclampsia: rationale for antioxidant clinical trials. Hypertension 44,374–380.
Rajan DP, Huang W, Dutta B, Devoe LD, Leibach FH, Ganapathy V and Prasad PD (1999) Human placental sodium-dependent vitamin C transporter (SVCT2): molecular cloning and transport function. Biochem Biophys Res Commun 262,762–768.[CrossRef][Web of Science][Medline]
Redman CW and Sargent IL (2005) Latest advances in understanding preeclampsia. Science 308,1592–1594.
Rice ME (2000) Ascorbate regulation and its neuroprotective role in the brain. Trends Neuroscience 23,209–216.[CrossRef][Web of Science][Medline]
Siiteri PK and Stites DP (1982) Immunologic and endocrine interrelationships in pregnancy. Biol Reprod 26,1–14.[Abstract]
Song J, Kwon O, Chen S, Daruwala R, Eck P, Park JB and Levine M (2002) Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2 (GLUT2) intestinal transporters for vitamin C and glucose. J Biol Chem 277,15252–15260.
Sotiriou S, Gispert S, Cheng J, Wang Y, Chen A, Hoogstraten-Miller S, Miller GF, Kwon O, Levine M, Guttentag SH et al. (2002) Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival. Nat Med 8,514–517.[CrossRef][Web of Science][Medline]
Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen X-Z, Wang Y, Brubaker RF and Hediger A (1999) A family of mammalian Na+-dependent L-ascorbic acid transporters. Nature 399,70–75.[CrossRef][Medline]
Vermillion ST and Robinson CJ (2005) Antiprostaglandin drugs. Obstet Gynecol Clin North Am 32,501–517.[CrossRef][Web of Science][Medline]
Vesce F, Farina A, Giorgetti M, Jorizzo G, Bianciotto A, Calabrese O and Mollica G (1997) Increased incidence of pre-eclampsia in pregnancies complicated by foetal malformation. Gynecol Obstet Invest 44,107–111.[Web of Science][Medline]
Xue S, Slater DM, Bennett PR and Myatt L (1996) Induction of both cytosolic phospholipase A2 and prostaglandin H synthase-2 by interleukin-1
in WISH cells is inhibited by dexamethasone. Prostaglandins 51,107–124.[Web of Science][Medline]
Submitted on July 20, 2006; resubmitted on October 2, 2006; accepted on October 9, 2006.
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