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Vol. 294, Issue 1, 141-146, July 2000
Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, University of Toronto, Toronto, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The inhibitory effects of cocaine and nicotine on placental amino acid transport, as a mechanism contributing to intrauterine growth restriction, were investigated in the in vitro placental perfusion model. Amino acids that represent substrates for known placental transporters were selected: alanine (system A), glutamine (system N), phenylalanine and valine (system l), and arginine (system y+). Amino acid accumulation on the fetal side was measured in the absence of cocaine or nicotine (n = 7) and in the presence of 1.2 µg/ml cocaine (n = 6), 120 ng/ml nicotine (n = 6), or both (n = 6). Neither cocaine nor nicotine alone significantly inhibited alanine transport, whereas their combination did (P = .02). Significant inhibition of arginine transport was detected with nicotine (P = .007), cocaine (P = .01), and their combination (P = .01), whereas phenylalanine (P = .03, P = .04) and valine (P = .03, P = .04) transport was affected by cocaine and the combination of cocaine and nicotine, respectively. For glutamine, neither cocaine, nicotine, nor their combination had a statistically significant inhibitory effect. In conclusion, both cocaine and nicotine may contribute to fetal growth restriction by interfering with the activity of amino acids transporters that are necessary to maintain the nutrient gradients associated with normal fetal growth.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cocaine
abuse is widespread, and it is often the drug of choice of a pregnant
substance abuser because of its availability, euphorigenic properties,
and addictive nature. The prevalence of fetal exposure to cocaine is
reported in various studies to range from 10 to 30% (Bateman et al.,
1993
; Koren, 1993; Eyler et al., 1998). Intrauterine growth restriction
(IUGR) is one of the consistently observed adverse perinatal outcomes
associated with cocaine use in pregnancy.
Cocaine users also have significantly greater exposure to other harmful
substances. Among cocaine users, more than 85% also smoke cigarettes,
compared with approximately 20% among the nonusers (Zuckerman et al.,
1989; Neuspiel et al., 1991). As with cocaine, IUGR is one of the most
prominent adverse effects associated with maternal cigarette smoking
during pregnancy. Some studies have suggested that the concurrent use
of cocaine with cigarette smoking may be more harmful to fetal growth
than either substance used independently (MacGregor et al., 1987;
Zuckerman et al., 1989). A study completed in Toronto by our group
documented that babies exposed to both cocaine and cigarette smoke had
a mean birth weight that was lower by 800 g, compared with
200 g lower for babies of "smoking only" mothers (Forman et
al., 1993).
In cocaine users, it is difficult to define and control other
potentially confounding factors on fetal growth such as maternal morbidity, poor maternal nutrition, or inadequate perinatal care. It is
even less well understood how IUGR associated with cocaine use in
pregnancy might be modified by the presence of cigarette smoking.
Nevertheless, available data indicate that IUGR associated with both
cocaine use and cigarette smoking in pregnancy may be a result of
combined effects of these compounds on fetal growth (Pastrakuljic
et al., 1999).
The relatively acute and transient nature of the vasoactive properties
of cocaine and cigarette smoke suggest that fetal growth restriction
associated with maternal cocaine use and cigarette smoking cannot be
attributed solely to their vasoconstrictive properties. Fetal growth
depends not only on nutrient provision via the uteroplacental
circulation but also on nutrient transport across the
syncytiotrophoblast. Studies in animals and in placental vesicles have
shown that cocaine and nicotine have the ability to inhibit amino acid
transport across the placenta. Therefore, they may directly affect the
transfer of amino acids, a mechanism which has been demonstrated in
IUGR pregnancies produced by agents other than cocaine and nicotine
(Varma and Ramakrishan, 1991; Pastrakuljic et al., 1999).
Cocaine and nicotine interaction with amino acid transport systems in
the syncytiotrophoblast may result in a deficit in the transport of
amino acids across the placenta. As a consequence, decreased or altered
amino acid pools may be available to the fetus. The lack of adequate
pools of essential amino acids especially can result in a marked
reduction in the rate of fetal growth.
Given the high rates of IUGR in newborns of cocaine users and cigarette smokers, in vitro evidence of amino acid transport inhibition by cocaine may provide the basis for a mechanistic understanding of how cocaine and other drugs of abuse might give rise to IUGR. Therefore, the aim of this study was to determine the inhibitory effect of cocaine, nicotine, and a combination of cocaine and nicotine on the net flux of five amino acids that represent different transport systems in human placenta: alanine (system A), valine and phenylalanine (system l), glutamine (system N), and arginine (system y+).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals. Nicotine hydrogen tartrate salt, (S)-nicotine, MgCl2, Hanks' balanced salt solution (HBSS), L-alanine, L-valine, L-arginine, L-phenylalanine, L-glutamine, 5-sulfosalicylic acid, heparin sodium salt, kanamycin, and glucose were obtained from Sigma (St. Louis, MO); KCl and trichloroacetic acid were obtained from BDH Inc. (Toronto, Ontario, Canada); Na2EDTA was obtained from Fisher Scientific (Orlando, FL); and cocaine was obtained from Radian International (Austin, TX). The radioimmunoassay (RIA) for nicotine was obtained from the Department of Biochemistry, Brandeis University (Waltham, MA), the RIA for cocaine/cocaine metabolite was obtained from Immunalysis (San Dimas, CA), and the RIA for human chorionic gonadotrophin (hCG) was obtained from Hybritech (San Diego, CA).
Study Design.
Placentae were obtained after vaginal or
cesarean section delivery from uncomplicated, term pregnancies and
transported to the laboratory in heparinized ice-cold PBS. Only
placentae from pregnancies with no maternal history of cigarette
smoking, chronic drug therapy, or drug/alcohol exposure were perfused.
Independent maternal and fetal circulations were established to a
peripheral placental lobule within 30 min of the delivery of the infant
as previously described by our laboratory (Simone et al., 1994). In
brief, a chorionic artery and vein supplying a peripheral placental lobule were selected and cannulated with flow rates approximately 13 to
15 ml/min on the maternal side and 3 to 4 ml/min on the fetal side.
Fetal inflow pressure was measured in the fetal arterial circuit
proximal to its insertion into the cannulated chorionic artery. The
fetal perfusate was recirculated through a 150-ml reservoir, and
maternal perfusate was recirculated through a 250-ml reservoir.
). Nicotine and cocaine
were measured by RIA assays.
The amino acid analyses were performed on an LKB 4145 Alpha Plus System
(LKB, Biochrom Limited, Cambridge, England) with norleucine as
an internal standard. The separation of the amino acids was accomplished using a 20-cm ion-exchange column inside a column heater.
A 20-µl sample containing a mixture of amino acids was loaded into
the column and mixed with the ninhydrin reagent, and the mixture was
passed through the high-temperature reaction coil. The colored
compounds produced from the reaction were detected at 570 nm. The
amount of colored compound produced was directly proportional to the
quantity of amino acid present in the eluate. The area under the peak
indicated the quantity of amino acid present. The coefficient of
variation was 1.5%.
The RIA procedure for nicotine used
[3H]nicotine, an antiserum raised in rabbits,
and a goat anti-rabbit gamma globulin (IgG) to separate the
antibody-bound nicotine or cotinine from the free analyte (Obach and
Van Vunakis, 1990). A mixture of 0.1 ml of [3H]nicotine, 0.1 ml of appropriately diluted
serum, 0.1 ml of an unknown sample or 0.1 ml of known standard
solution, and isogel Tris buffer (0.15 M NaCl, 0.01 M Tris,
0.1% gelatin, pH 7.4) for a total volume of 0.8 ml was incubated for
1 h at 37°C. The double antibody technique was used to separate
bound and free [3H]nicotine by adding 0.1 ml of
normal rabbit serum and 0.1 ml of goat anti-rabbit IgG serum at
appropriate dilutions and incubating overnight at 4°C. After
centrifugation at 1000g, 4°C, for 45 min, the
immunoprecipitates were dissolved in 0.1 M NaOH (0.1 ml), and
radioactivity was determined after addition of 2.5 ml of scintillation fluid. The radioactivity of the sample was determined using a scintillation counter and was expressed as counts per minute. The
technique was sensitive to 0.5 ng/ml.
The RIA procedure for cocaine was performed as follows: 25 µl of
unknown specimen, standard specimen, or normal control was mixed in
test tube with a 100-µl dilution of sheep anti-cocaine antibody
(polyclonal) and 200 µl of a radiolabeled antigen,
125I-benzoylecgonine (Simone et al., 1994). After
precipitation with 200 µl of secondary antibody complex and
incubation for 2 h at room temperature, samples were centrifuged
for 30 min at 2000g. The tubes were decanted, and the
pellets containing bound antigen were counted in a gamma scintillation
counter; results were expressed as counts per minute. The technique was
sensitive to 2 ng/ml, and the dose-response curve was used for
calculation of the concentration of unknown specimens.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The preliminary time course studies, with HBSS as a perfusion medium, showed that the placental preparation retained physical integrity and optimal metabolic activity for up to 2.5 h in a closed circuit experimental design. Accordingly, a time period of 140 min was adopted for the amino acid transport studies. Amino acid transport studies in the dually perfused placenta were conducted under four separate experimental conditions: control group, cocaine group, nicotine group, and cocaine and nicotine group. The data for amino acid levels in the fetal and maternal circulations were expressed as fetal/maternal (F/M) concentration ratios. To ensure that placentae used in experiments were naive to nicotine and cocaine, cocaine, nicotine, and cotinine levels were measured in the placental tissue of all placentae before perfusion. None of the placental tissue tested had detectable levels of either cocaine, nicotine, or cotinine before perfusion.
The mean mass of the perfused cotyledons, the fetal inflow pressure,
and the fetal and maternal flow rates are shown in Table 1. Placental glucose and oxygen
consumption and lactate production, as indicators of metabolic
viability of the placental lobule (Table 1), did not significantly
differ between control and experimental groups (Table
2). The preferential secretion of hCG
into the maternal circulation was also verified (Table 1) and was not significantly different between control and experimental groups (Table
2).
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Using seven placentae in the control group, the transfer of alanine,
glutamine, phenylalanine, valine, and arginine toward the fetal
circulation increased as a function of time during the observation
period of 140 min. As shown in Table 3,
the highest accumulation after 140 min on the fetal side of 33 ± 7.6% was observed for arginine. Under control conditions,
phenylalanine and valine showed similar accumulations of 13 ± 5 and 13 ± 4%, respectively. Alanine showed an accumulation of
9 ± 1.5%, whereas glutamine showed the lowest accumulation of
7 ± 10%.
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In the cocaine group (six placentae), the initial concentrations of cocaine in the maternal and fetal reservoirs were 1.12 ± 0.05 and 1.12 ± 0.15 µg/ml, respectively. In the group of six placentae perfused in the presence of nicotine alone, the initial concentration of nicotine in the maternal reservoir was 119 ± 7 and 118 ± 5 ng/ml in the fetal reservoir. In the group of six placentae perfused with both cocaine and nicotine, the concentration of cocaine in the maternal and fetal reservoirs was 1.2 ± 0.09 and 1.3 ± 0.07 µg/ml, respectively, and the concentration of nicotine in the maternal and fetal reservoirs was 113 ± 17 and 117 ± 20 ng/ml, respectively.
The cocaine and nicotine concentrations used in the in vitro perfusion studies were approximately 3 times higher than that observed in in vivo users. This was done to ensure that the effect of cocaine and nicotine on transplacental amino acid transport could be observed within the time frame of the experiment, which is much shorter relative to the in vivo exposure over the entire course of the pregnancy.
As illustrated in Fig. 1, alanine
transport toward the fetal circulation was not significantly reduced in
the presence of either cocaine alone or nicotine alone. However, the
combination of cocaine and nicotine significantly inhibited alanine
transport (Table 3). Alanine was the only amino acid that showed a
lower accumulation in the presence of both drugs than in the presence of each drug alone.
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For glutamine (Fig. 2), neither cocaine,
nicotine, nor their combination had a statistically significant
inhibitory effect on its accumulation on the fetal side of the
placenta. Although not statistically significant, the numerical value
for the F/M ratio decreased in all three experimental groups,
suggesting a reduction in glutamine transport (Table 3).
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The F/M ratios of phenylalanine and valine in control and experimental
groups are shown in Figs. 3 and
4, respectively. Cocaine alone
significantly reduced phenylalanine and valine transport compared with
the controls. In contrast, nicotine alone had no statistically
significant inhibitory effect, although there was an apparent decrease
in transport of both amino acids. A statistically significant reduction
in the transport of both amino acids was also observed in the presence
of both cocaine and nicotine, but this reduction is likely to be
independent of a nicotine effect (Table 3).
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Statistically significant inhibition of arginine transport (Fig.
5) was detected in all three experimental
groups with a prominent nicotine effect. However, an additive or
synergistic inhibitory effect on arginine accumulation on the fetal
side was not observed with the combination of cocaine and nicotine
(Table 3).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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By dually perfusing the placental cotyledon in vitro, the effects of cocaine and nicotine on the transport of amino acids across the human placenta as a mechanism contributing to IUGR seen clinically was investigated. Our hypothesis was based on epidemiological evidence that cocaine use concurrent with cigarette smoking in pregnancy may result in even more serious IUGR than either substance used independently. At the present time, the potential combined effect of cocaine and cigarette smoking on fetal growth through their effects on placental amino acid transport has not been elucidated.
Alanine, a system A substrate, was selected for the study for two
reasons. First, system A is the dominant transport system in the
microvillous membrane of the syncytiotrophoblast that transports neutral amino acids. Second, given that alanine has the highest concentration of all amino acids in the human plasma and is a major
fetal gluconeogenic substrate, inhibition of alanine transport toward
the fetal circulation may have serious implications on fetal growth
(Pastrakuljic et al., 1999).
In the presence of both cocaine and nicotine, the alanine F/M
concentration ratio was significantly decreased, suggesting a combined
effect of cocaine and nicotine on the system A transporter. Therefore,
this mechanism may play a critical role in more serious IUGR in
pregnancies exposed to both drugs. Although statistically not
significant, an apparent trend of decreased alanine transport was
evident with both cocaine alone and nicotine alone. If a greater number
of experiments was available to confirm that this trend is significant,
it could be of clinical significance. For example, even a small
decrease in alanine transport could have a profound effect on fetal
growth when exposure is long-term over the course of gestation. The
observations that cocaine and nicotine alone could have the potential
to inhibit alanine transport and affect the system A transporter are
consistent with published data. Cocaine was shown to inhibit alanine
transport when added in vitro to microvillous and basal membrane
vesicles derived from human placenta, whereas nicotine was shown to
inhibit uptake of system A substrates in human placental slices
(Barnwell and Sastry, 1983; Dicke et al., 1993, 1994). In contrast, the
study by Krishna et al. (1995) showed that concentrations of cocaine
likely to be encountered in vivo do not affect alanine transport across
the perfused human placenta. A possible explanation for these negative
findings is that the placental tissue was exposed to cocaine for only a
short period of time (40 min).
The potential adverse impact of cocaine and nicotine on fetal growth
was also explored by testing their effects on arginine transport across
the placenta. Arginine is primarily transported by system
y+, and given its high capacity, system
y+ is believed to serve as a major cationic
transporter on both membranes of the syncytiotrophoblast (Moe, 1995).
In addition, the cationic amino acids, such as arginine, have
physiological uptakes that barely exceed their accretion rates into
fetal proteins. Under normal circumstances, there is a very narrow
margin between placental arginine transport capacity and fetal demand,
implying that even slight alterations in transporter activity due to
exposure to cocaine and nicotine may result in fetal growth restriction.
In this study, a significant decrease in the F/M concentration gradient of arginine was observed with cocaine and with nicotine alone, as well as with their combination. Figure 5 provides evidence that there is no additional inhibition of arginine transport when both cocaine and nicotine were present, suggesting a lack of combined effect. These observations suggest that one of the possible mechanisms by which cigarette smoking and cocaine affect fetal growth may be impairment of system y+.
These finding are in contrast with studies in rat placental vesicles
that showed no effect of cocaine on arginine transport (Novak et al.,
1995). This could be due to species differences that are likely to
exist between the human and the rat placenta with regard to amino acid
transporters. There are no studies of cocaine or nicotine effect using
human placental vesicles. However, a study with lysine, another system
y+ substrate, showed that high concentrations of
cocaine have the potential to inhibit lysine transport in human
placental villous fragments (Barnwell and Sastry, 1983), which is in
agreement with our findings. Cordocentesis studies in humans also
suggest that arginine transport may be significantly depressed in IUGR
pregnancies not resulting from cocaine use (Cetin et al., 1996).
Phenylalanine and valine showed a similar pattern of transfer toward
the fetal circulation in control and experimental groups of placentae.
Both amino acids are transported by sodium-independent system l,
located on both membranes of the syncytiotrophoblast. In the presence
of nicotine alone, no significant inhibition of phenylalanine and
valine transport toward the fetal circulation was observed. The F/M
concentration ratio showed a statistically significant inhibitory
effect of cocaine on the transport of phenylalanine and valine,
suggesting that cocaine is a major factor affecting system l when both
drugs are used together. Regarding the fact that system l is also
responsible for sodium-independent transport of alanine, inhibition of
this system by cocaine may significantly contribute to inhibition of
alanine transport as well. In addition, the significance of this
reduction for fetal growth is that branched-chain amino acids are
extensively used by the fetus for energy and protein synthesis, and
therefore an impairment of their transplacental transport may result in
fetal growth restriction. This hypothesis is confirmed by studies in
IUGR pregnancies that consistently show low fetal levels of
branched-chain amino acids (Cetin and Pardi, 1994).
For glutamine, a system N substrate, neither cocaine, nicotine, nor their combination had a statistically significant inhibitory effect on its transport toward the fetal circulation. A trend toward a decrease in the F/M concentration ratio in the presence of cocaine and nicotine alone and their combination was evident. However, this apparent inhibition of glutamine transfer lacked statistical significance, possibly due to the limitations of detecting differences with the low rates of glutamine transfer observed in the control group of placentae. The importance of system N inhibition by cocaine is not only in the context of reduced glutamine transport but also on the effect this has on glutamate synthesis. Present findings suggest that glutamate synthesis is not likely to be affected in drug-exposed fetuses.
Both cocaine and nicotine are potent vasoconstrictors, and their vascular effects could also result in reduced placental nutrient transfer to the fetus. Findings from this study clearly show that vasoconstriction is not the only cause of amino acid uptake and transfer inhibition. However, when these drugs are abused together, vasoconstriction may act to exacerbate other causes of decreased amino acid transfer to the fetus such as drug interaction with amino acid transporters.
In conclusion, the in vitro placental perfusion studies showed a significant relationship between cocaine and decreased amino acid transport of phenylalanine (system l) and arginine (system y+) and between nicotine and decreased amino acid transport of arginine (system y+). Regarding their combined drug effect, cocaine and nicotine appeared to have a combined inhibitory effect on alanine (system A). Furthermore, cocaine and nicotine alone showed a trend to decrease transport of all five amino acids tested, suggesting that their continuous presence throughout pregnancy may result in fetal amino acid deprivation, further resulting in fetal growth restriction. Further studies are needed to corroborate these findings with in vivo measurements of maternal/fetal and maternal/neonatal concentration ratios of amino acids in growth restricted infants exposed in utero to cocaine, cigarettes, and their combination.
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Footnotes |
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Accepted for publication March 17, 2000.
Received for publication January 5, 2000.
1 This work was supported by a grant from the Medical Research Council of Canada.
Send reprint requests to: Dr. Gideon Koren, Department of Clinical Pharmacology (rm. 8239), Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. E-mail: placgrp{at}sickkids.on.ca
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Abbreviations |
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IUGR, intrauterine growth restriction; F/M, fetal/maternal; RIA, radioimmunoassay; hCG, human chorionic gonadotrophin; HBSS, Hanks' balanced salt solution.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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