Transfer of cocaine and benzoylecgonine across the perfused human placental cotyledon

BASIC SCIENCE SECTION

Transfer of cocaine and benzoylecgonine across the perfused human placental cotyledon Carmine Simone, BSc"' Lidia O. Derewlany, PhD," Marilynne Oskamp, BScN,d Brenda Knie, RT," and Gideon Koren, MD" b, C

C

Toronto, OntarIO, Canada OBJECTIVE: Our aim was to measure the transfer of cocaine and its major metabolite benzoylecgonine across the human term placenta. STUDY DESIGN: By means of in vitro perfusion of the human term placental cotyledon the transfer of these compounds was measured. RESULTS: The steady-state maternal-to-fetal transfer of cocaine (0.18 ± 0.05 j.Lg/ml/min) was significantly greater than benzoylecgonine transfer (0.02 ± 0.01 j.Lg/ml/min) (p <: 0.05). When the perfused tissue was analyzed 32% ± 7% of the maternal cocaine dose was retained by the placental tissue, whereas only 12% ± 12% of the maternal benzoylecgonine dose was retained by the placental compartment. CONCLUSIONS: These results suggest (1) the placenta may serve as a depot for large amounts of cocaine, thus offering some degree of fetal protection after bolus administration; (2) fetal exposure may be prolonged by placental retention and subsequent release of cocaine and benzoylecgonine; and (3) benzoylecgonine does not cross the placenta as readily as does cocaine. Variability in placental handling of cocaine and benzoyl~cgonine may therefore determine fetal exposure to these agents. (AM J OSSTET GVNECOL 1994;170:1404-10.)

Key words: Cocaine, benzoylecgonine, placenta, in vitro perfusion For decades it was assumed that the human placenta serves as a barrier, protecting the fetus from exposure to xenobiotics circulating in the maternal system. The thalidomide disaster three decades ago completely reversed this concept. It has become apparent that the placenta allows most xenobiotics and their metabolites to cross from the maternal to the fetal side. Although the number of proved teratogens is limited, hundreds of compounds have been documented to exert a direct pharmacologic or toxic effect on the fetus when taken by the mother. I Cocaine is an example of such a compound. Previous studies have revealed cocaine use in pregnancy to be associated with a high rate of spontaneous abortion, abruptio placentae, prematurity, and intraFrom the DIViSIOn of Clmieal Pharmacology and ToxlCologya and the MotheRllk Clnlle: Department of Pharmacology, Umvers!t.~ of Toronto,' and the DiVISIOn of Obstetncs and Gynecology, Mt. Smai Hospltal d Supported by a grant from the Medical Research Counetl of Canada. C.S. IS supported by a studentshlp from the Medtcal Research Counnl of Canada. G.K. IS a Career SClentllt with the Ontano Mlnlltry of Health. Receitlfd for publzcatlOll May 11, 1993; reVIsed October 1, 1993; accepted November 12, 1993. Reprmt leque.lts: (;uleoll Amen, AIV, VlVl.IWIl oj UlIllwl l'lwJ 1Ilt/eology and TOXicology, Hosp!tal for Sick Chzldren, 555 UnlVemty Ave., Toronto, Ontario, Canada M5G 1X8. Copyright © 1994 by Mosb_~-Year Book, Inc 0002-9378/94 $3.00 + 0 6/1/52862

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uterine growth retardation."' 3 It has also been suggested that cocaine may have a teratogenic effect on the human fetus!-? Recently our laboratory has demonstrated that approximately 12% of babies born in a downtown Toronto hospital are exposed to cocaine during the last trimester of gestation. In American inner-city centers the figures are higher, with approximately 17% of babies being exposed to cocaine in utero. s , q Therefore the effect of cocaine used by pregnant women on their developing fetuses has become a topic of major public health importance. The elimination half-life of cocaine is relatively short (20 to 90 minutes), but some of the consequences of its use, such as seizures and strokes, can occur hours after exposure." O A variety of clinical seizure types are described in human newborns exposed to cocaine prenatally.1O However, approximately half the seizures in newborns with prenatal cocaine exposure are resolved in < 3 weeks after birth."o These observations suggest a long-lasting pharmacologically active agent capable of inducing such seizures may persist long after the disappearance of cocaine.!! The m
Volume 170, Number 5, Part 1 Am J Obstet Gynecol

cocaine on isolated perfused cerebral arteries.!3-!5 These observations suggest that benzoylecgonine may cause some of the cocaine-related adverse perinatal effects. The placental transfer of cocaine and benzoylecgonine is of great practical interest to the clinician trying to relate exposure to clinical outcome. There are several animal studies addressing placental transfer and fetal tissue uptake of cocaine and benzoylecgonine!6-!9; however, extrapolation to human pregnancy is difficult. Although the transfer kinetics of cocaine across the human placenta perfused in vitro has been recently measured, there has been no study investigating the transfer of benzoylecgonine across the human placenta. 20 Because of ethical and safety concerns for the unborn child and mother, research in this area is understandably lacking. The current study measures the placental transfer of cocaine and its major metabolite benzoylecgonine by means of the in vitro perfusion of the human placental cotyledon. An understanding of the pharmacokinetics of cocaine transfer by means of this model will be useful in relating cocaine exposure to its pharmacologic effects in vivo. Material and methods

Perfusion technique. Term placentas were obtained after delivery by cesarean section and were transported to the laboratory in ice-cold heparinized saline solution. Only placentas from normal, uncomplicated term pregnancies with no maternal history of clinically significant infectious diseases were perfused. Independent maternal and fetal circulations were established to a peripheral placental lobule as previously described by our laboratory.2! A chorionic artery and vein supplying a single peripheral lobule were selected and cannulated with flow rates approximating 13 to 15 ml/min on the maternal side and 4 to 5 ml/min on the fetal side. In "closed" experiments the fetal perfusate was recirculating through 150 ml reservoir and the maternal perfusate through a 250 ml reservoir. In "open" experiments both fetal and maternal perfusates were not recirculated, and fresh buffered perfusate was allowed to perfuse the isolated cotyledon. Perfusion of the placenta was established within 30 to 45 minutes of delivery of the infant. The perfusate (maintained at 37° C) consisted of a tissue culture medium (MI99, Sigma, St. Louis) that contained heparin (2000 U/L), kanamycin (100 mg/L), glucose (1/0 gm/L), and 40,000 molecular weight dextran (fetal 30 gm/L, maternal 7.5 gm/L). Sodium bicarbonate was added to maintain the pH of the perfusates within a physiologic range (fetal pH 7.35, maternal pH 7.40) throughout the experiment. The fetal perfusate was equilibrated with 95% nitrogen and 5% carbon dioxide, whereas the maternal perfusate was equili-

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brated with 95% oxygen and 5% carbon dioxide. After residual blood was cleared out of the fetal vessels and the maternal intervillous space, the maternal and fetal circuits were recirculated. A 2-hour closed-circuit control period preceded the experimental period. During the control period parameters such as glucose and oxygen consumption and lactate and human chorionic gonadotropin (hCG) production were determined, to establish baseline values for each experiment. Also, the integrity of the preparation was assessed by monitoring the stability of the fetal perfusion pressure (50 to 60 mm Hg) and measuring the volume loss from the fetal circulation. Preparations that showed a volume loss > 3 ml/hr indicated a leak of perfusate from the fetal to the maternal compartment and were not included in the study. After the 2-hour control period the perfusates in both the maternal and fetal circulations were replaced with fresh media. At this time the maternal perfusate also contained 0.60 ± 0.05 j..lg/ml of "crack" (alkaloidal) cocaine or 0.65 ± 0.05 j..lg/ml benzoylecgonine (Health Protection Branch, Health and Welfare Canada). Arterial and venous samples (open circuit) or reservoir samples (closed circuit) were obtained from both the fetal and maternal circuits every 5 minutes for the first half hour and every half hour subsequently, to measure cocaine concentrations. Samples of perfusate were obtained from the maternal and fetal reservoirs at 30minute intervals for measurement of hCG concentration, lactate production, and glucose consumption. Additional arterial and venous samples were taken throughout the perfusion for measurement of pH, Po 2 , and Pco 2 with a blood gas analyzer (Radiometer ABL 330, Copenhagen). At the end of the 2-hour experimental period the perfusates in both the maternal and fetal circulations were once again replaced with fresh media. This period is referred to as the postexperimental control period. Once again, reservoir samples were obtained for measurement of cocaine, glucose, lactate, and hCG. Both circuits were recirculated, and all parameters measured were compared with the initial control period to ensure the maintained integrity of the preparation. Some of the experiments (n = 3 for both benzoylecgonine and cocaine) included a I-hour open circuit perfusion after the 2-hour closed-circuit experimental period. The perfusates in both maternal and fetal compartments were replaced with fresh perfusate. The maternal perfusate for the open-circuit perfusions contained 0.60 j..lg/ml cocaine or 0.60 j..lg/ml benzoylecgonine. Both circulations were not recirculated, and the venous effluent was discarded. Fetal artery and vein and maternal fetal and vein samples were collected, and drug concentrations were determined as outlined below.

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When the experiment was terminated, samples of both perfused and nonperfused tissue were collected to determine tissue binding of cocaine. Sample analysis. Perfusate samples were kept at -70 C until the day they were analyzed. Cocaine and benzoylecgonine were measured by means of commercially available radioimmunoassays. Cocaine concentrations were determined with the Coat-A-Count RIA for Cocaine Metabolite (Diagnostic Products, Los Angeles) using in-house "crack" standards. It has been previously determined that although this radioimmunoassay was designed for benzoylecgonine measurement, the great cross-reactivity (> 100%) with cocaine by the primary antibody allows for it to be used for accurate quantitative cocaine detection. 22. 23 Cross-reactivity with benzoylecgonine is only 0.5% and with ecgonine methyl ester 6.0%. The sensitivity of the assay is 0.5 ng/mI, which corresponds to 0.1 I-lg/kg tissue. Benzoylecgonine was measured with the Abuscreen RIA for Cocaine Metabolite (Hoffman LaRoche, Nutley, N.J.). The crossreactivity with cocaine was found to be 4%, and there was no cross-reactivity with ecgonine methyl ester. The sensitivity of the assay i~ 5 ng/ml, which corresponds to 1.0 I-lg/kg tissue. At the end of each experiment samples of perfused and nonperfused placental tissue were assayed for the presence of cocaine and benzoylecgonine with the above mentioned radioimmunoassasys. The cocaine and benzoylecgonine were extracted from the placental tissue by means of the method previously described by our laboratory. 22 Lactate concentrations were determined by measuring the reduction of nicotinamide adenine dinucleotide by lactate dehydrogenase (Sigma lactate procedure No. 826-UV). Glucose concentrations were measured with a YSI 23A glucose analyzer. Perfusate concentrations of hCG were measured by time-resolved fluorescence immunoassay with EuroFluor-S on a CyberFluor 615 Immunoanalyzer (CyberFluor Inc., Toronto). The ability of the placental tissue to synthesize hCG and to secrete it preferentially into the maternal circulation during perfusion further quantified the viability and physical intactness of these placental preparations. Statistics. A Student t test was used for comparison of the rates of maternal-to-fetal transfer of the compounds and for comparison between cocaine and benzoylecgonine tissue retention. Differences were considered significant at a level of p < 0.05. All data are reported as the mean ± 1 SD. 0

Results

Physical parameters. The mean mass of the perfused cotyledons was 14.32 ± 4.54 gm (n = 8). There was no significant difference in the fetal arterial inflow pressures between the control and experimental periods; the pressures were 58 ± 3 mm Hg (n = 8) for

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the control period, 60 ± 6 mm Hg (n = 8) for the experimental period, and 59 ± 5 mm Hg (n = 6) for the postexperimental period. The fetal flow rate was 4.5 ± 0.2 mllmin and the maternal flow rate 17.0 ± 0.6 mllmin for all the experiments. Viability and physical integrity. Measurements of placental lactate production and oxygen and glucose consumption indicate metabolic viability of the placental cotyledon throughout the perfusion setup. Values for these parameters did not vary throughout the course of the perfusion experiments. The appearance of hCG only in the maternal circulation and not in the fetal circulation reflects the expected preferential secretion of the hormone and indicates physical integrity of the placental preparation. Closed-circuit experiments (n = 4). Cocaine or benzoylecgonine was added to the maternal circulation at concentrations previously reported in the plasma of cocaine users (i.e., 0.60 ± 0.05 I-lg/ml).24 The rate of cocaine disappearance from the maternal circulation and the rate of appearance in the fetal circulation was calculated by means of the slope of the cocaine (in micrograms) versus time (in minutes) curve. The slope was calculated by applying linear regression to the linear portion of the curve (the first 20 minutes). The rate of cocaine disappearance was 1.99 ± 0.56 I-lg/min from the maternal circulation. The rate of cocaine appearance in the fetal circulation was 0.66 ± 0.07 I-lg/min. Both fetal and maternal circulations equilibrated within approximately 100 minutes of the start of the experiment (Fig. 1) at a concentration approximately 32% of the initial concentration in the maternal circuit (i.e., 0.19 I-lg/ml). The rate of benzoylecgonine disappearance from the maternal circulation was 0.45 ± 0.15 I-lg/min. The rate of benzoylecgonine appearance in the fetal circulation (0.15 ± 0.05 I-lg/min) was significantly lower than the rate of cocaine appearance (p < 0.05). Open-circuit experiments (n 3). With an open circulation on both sides of the placenta, steady-state transfer was investigated. Cocaine transfer was 0.18 I-lg/min/gm tissue. The rate of benzoylecgonine steadystate transfer was 0.02 ± 0.01 I-lg/min/gm tissue (Table I), which is significantly less than that of cocaine (p < 0.05). Postexperimental control period (n 3). Mter replacement of the fetal and maternal perfusates after the cocaine perfusion experiments, the fetal and maternal circulations achieved final concentrations of 0.10 ± 0.02 I-lg/ml and 0.09 ± 0.02 I-lg/ml, -respectively, after 60 minutes (Fig. 2). Mter the benzoylecgonine perfusion experiments the final fetal concentration of benzoylecgonine was 0.04 ± 0.0 I I-lg/ml, whereas the maternal concentration was 0.10 ± 0.04 I-lg/ml after 60 minutes (Fig. 2). Tissue retention (n = 3). Mass balance studies per-

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Fig. 1. a, Time course of cocaine disappearance from maternal circulation (filled circles) and appearance in fetal circulation (open circles); b, time course of benzoylecgonine disappearance from maternal circulation (filled circles) and appearance in fetal circulation (open circles). Data obtained from closed-circuit perfusions of human placental cotyledons (n = 4 for both compounds).

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Fig. 2. Mter 2-hour experimental period when cocaine or benzoylecgonine was perfused on maternal side, both maternal and fetal perfusates were replaced with fresh drug-free perfusate, and leaching out from placental compartment was investigated. a, Time course of cocaine appearance in maternal (filled Circles) and fetal (open czrcles) circulation during postexperimental period; b, time course for benzoylecgonine appearance.

Table I. Rate of maternal-to-fetal transfer of benzylecgonine and cocaine across perfused placental cotyledon Expenment 1

2

3 Mean ± SD

Rate of transfer of cocazne (f.1f5/min/gm)

Rate of transfer of benzo.vlecgonme (f.1f5/mm/gm)

0.22 O.IS 0.13 0.IS±0.05

0.01 0.02 0.02 0.02 ± 0.01*

*Transfer rates are signifi<.antly different (p < 0.05).

formed at the end of the perfusion experiments show that after 120 minutes of perfusion with cocaine in the maternal circuit 18% ± 1% of the maternal dose of cocaine crossed the placenta to the fetal side, whereas 32% ± 7% was retained by the placental compartment

(Table II). Mter 120 minutes of perfusion with benzoylecgonine in the maternal circuit, 8% ± 1% of the maternal dose crossed to the fetal side, whereas 12% ± 12% was retained by the placental compartment (Table III).

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Table II. Cocaine tissue retention

Experiment

1 2 3 Mean ± SD

Percent of total

Total amount of cocazne admimstered (pg)

Amount of cocame appearing zn fetal ClrCUlt (pg)

Amount of cocame zn perfused tISsue (pg)

Amount In ammotlc flUId (pg)

155 163 148 155 ± 8 100

29 30 27 29 ± 1 18 ± 1

57 40 53 50 ± 9 32 ± 7

5 20

ND

8 ± 10 5±6

Amount of cocaine remammg in maternal ClrcUlt (pg)

57 45 55 52 ± 6 34 ± 5

Recovery (%)

95 83 91 90 ± 6

ND, Not detected in sample saved from experiment.

Table III. Benzoylecgonine tissue retention

Expenment 1

2 3 Mean ± SD

Percent of total

Amount of Amount of Amount of Amount of Total amount benzoylecgonine benzoylecgonine benzoylecgonine benzoylecgonzne in amniotic remazning in maternal of benzoylecgonzne appeanng in fetal czrCUlt in perjuJed fluid (pg) administered (pg) (pg) tissue (pg) ClrcUlt (pg) Recovery (%)

175 168 175 173 ± 4 100

14 16 12 14 ± 2 8± 1

43

ND

20 21 ± 22 12 ± 12

5±8 3± 5

ND

14

ND

130 122 122 125 ± 5 72 ± 2

107 91 88 95 ± 10

ND, Not detected'.

Comment Although the term placenta is often regarded as a senescent organ, there is no evidence to support this commonly held view. 25 Even at term new villi are being formed, and time-related biochemical and morphologic changes can be attributed to growth and differentiation. 26 • 27 Studies comparing human early versus term placentas suggest that uptake and transport processes are similar in term placentas compared with those taken at earlier stages of gestation, although the capacity of the transport processes studied were not identicaF" Such findings are consistent with changes in placental function needed to meet changing fetal requirements throughout the course of gestation. Although the term placenta may differ on a quantitative basis from placentas at other stages of gestation, qualitative aspects are similar. The in vitro perfusion of the human placenta therefore is a good model for the in vivo transfer of compounds in human gestation. 21 • 29 To our knowledge, this study is the first to measure the transfer of benzoylecgonine across the perfused human placenta. Recently Schenker et a1. have shown that cocaine easily crosses the human placenta perfused in vitro. 20 Cocaine was observed to have a rate of transfer similar to that of antipyrine. In vivo animal studies have also shown cocaine to freely cross the placenta. Wiggins et a1. 16 found that during the period from 30 to 90 minutes after the administration of cocaine to pregnant dams cocaine appeared in fetal

brain at a rate of 109% to 150% of the concentration in the dam's blood. Spear et aI., 19 however, found that fetal concentrations of cocaine in brain and plasma were approximately twofold to threefold less than those of the dam's. Only anecdotal reports exist regarding cocaine and benzoylecgonine transfer across the human placenta in vivo. For example, Mittleman et a1. 24 report a case study in which a postmortem examination was performed on a pregnant woman who had self-administered a possible fatal dose of cocaine and subsequently died in a motor vehicle accident. In this case the maternal/fetal cocaine concentration ratio, when blood was compared, was 10: 1. The possible explanation offered by the authors for the discrepancy between fetal and maternal concentrations was incomplete maternal/fetal equilibration. We measured the rate of cocaine transfer across the in vitro perfused human cotyledon; it was 0.18 ± 0.05 f,Lg/min/gm tissue-approximately 10 times that of benzoylecgonine transfer. It is therefore apparent that the placenta does not provide a significant barrier to the transfer of cocaine from the maternal to the fetal circulations. The rapid transport of cocaine across the placenta can be explained by cocaine's low molecular weight (molecular weight 305) and high lipid solubility. Furthermore, cocaine is a weak base and is only 8% to 10% protein bound in human plasma. 8 Therefore the majority of the administered dose is available to equilibrate with the fetal circulation. As illustrated in Fig. 1, a,

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this equilibration can occur within 100 minutes, with cocaine being detected in the fetal circulation within 5 minutes of administration. Increasing the concentration of cocaine on the maternal side should not affect the maternal-to-fetal transfer measures obtained unless either carrier-mediated or active-transport processes were involved. 30 There is no evidence to date to support such a hypothesis. Recently benzoylecgonine which was thought to be a pharmacologically inactive metabolite of cocaine, has been shown to have a greater contractile effect than cocaine on isolated cerebral arteries from lamb and cat. I '. 14 Benzoylecgonine has also been shown to cause cerebral vasoconstriction in anesthesized piglets. 15 Given that benzoylecgonine has a much longer half-life than does cocaine, this compound may be of toxicologic importance in fetal neurodevelopment. The rate of benzoylecgonine transfer across the in vitro perfused human cotyledon was significantly less than that observed for cocaine. Furthermore, benzoylecgonine was not detected in the fetal circulation until approximately 15 minutes after its introduction into the maternal compartment. This can be explained in part by the greater hydrophilicity of benzoylecgonine compared with the very lipophilic cocaine molecule. The placenta therefore acts as a more effective barrier to benzoylecgonine transfer to the fetal circulation. Cocaine-binding protein has been previously described in the placenta by Ahmed et al.,31 yet the significance of this finding was not discussed by the authors. Our studies show that a significant amount of the maternal dose of cocaine and benzoylecgonine is retained by the placenta, which later leaches out into both fetal and maternal circulations, as seen in the postexperimental control period (Fig. 2). This may be significant in the fetal toxicity of cocaine and benzoylecgonine, where the effects of both compounds may be prolonged by retention and subsequent release by the placental compartment. Schenker et al. 20 did not comment on the significance of placental cocaine retention. Although substantial amounts of cocaine were measured in the placental tissue, it was not thought to be of importance by Schenker et al. Cocaine is most commonly self-administered intermittently, and thus the placenta may be exposed to boluses of cocaine as opposed to a steady plasma concentration. The placenta may modulate a bolus through its ability to retain different amounts of the administered dose of cocaine (32% ± 7%) (Table II). Placentas may differ substantially in their ability to serve as a depot and thus act as a buffer for the cocaine bolus. More experiments are needed under different conditions of bolus administration to observe a possible variation in placental binding capacity of cocaine.

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REFERENCES 1. Koren G. Teratogenic drugs and chemica". In: KOJen G. ed. Maternal-fetal toxicology. New York: Marcel Dekker, 1989: 15-28. 2. ChasnoffIJ, Burns WJ, Schnoll SH, Burns KA. Cocaine use in pregnancy. N EnglJ Med 1985;313:666-9. 3. MacGregor SN, Keith IJ, Chasnoff U. et al. Cocaine use and pregnancy: adverse perinatal outcome. A,\1 J OBSIET GVNECOL 1987:157:686-90. 4. Bingol N, Fuchs M. Diaz V, Stone RK, Gromlsch DS. Teratogenicity of cocaine in humans. J Pediatr 1987; 110: 93-6. 5. Chasnoff IJ, Chisum G. Kaplan W. Maternal cocaine use and genitourinary tract malformations. Teratology 1988; 37:201-4. 6. Fantel A, Macphail B. The teratogenicity of cocaine. Teratology 1982;26:17-9. 7. Hoyme HE, Jones KL, Dixon SD, et al. Prenatal cocaine exposure and fetal vascular disruption. Pediatrics 1990;85: 743-7. 8. Gmgras J, Mayer R, Hume R, O'Donnell K. Cocaine and development: mechanisms of fetal toxicity and neonatal consequences of prenatal cocaine exposure. Early Hum Dev 1992;31: 1-24. 9. Volpe J. Effect of cocaine use on the fetus. N EnglJ Med 1992;327:399-407. 10. Kramer LD, Locke Ge, Ogunyemi A, Nelson L. Neonatal cocaine-related seizures . .l Child Neurol 1990;5:60-4. 11. Konkol~, Erickson BA, Doerr JK, Hoffman RG, Madden JA. Seizures induced by the cocaine metabolite benzoylecgonine in rats. Epilepsia 1992;33:420-7. 12. Ripple M, Goldberger B, Caplan Y, Blitzer M, SchwartL S. Detection of cocaine and its metabolites in human amniotic fluid. J Anal Toxicol 1992;16:328-31. 13. Schreiber M, Torgerson L, Covert R, Madden J. Cocaine and metabolite-induced vasoconstriction of isolated pressurized cerebral arteries from penniltallambs. Pediatr Res 1992;21:221A. 14. Madden .l, Powers R. Effect of intraluminally infmed cocaine and metabolites on cat cerebral arteries in vitro. Life SCI 1990;47:1109-14. 15. Albuquerque M, Kurth C, Monitto C, Shaw L, Anday E. Cocaine metabolites affect cerebrovascular tone in newborn piglets. Pediatr Res 1992;31:57A. 16. Wiggins RC. Rolsten C, RUlZ B, Davis CM. Pharmacokinetics of cocaine: basic studies of route, dosage, pregnancy and lactation. Neurotoxicology 1989;10:367-82. 17. Sandberg .lA, Olsen GD. Cocaine and metabolite concentrations in the fetal guinea pig after chorionic maternal cocaine admmistration . .I Pharmacol Exp Ther 1992;260: 587-91. 18. DeVane CL. Burchfield DJ, Abrams RM, Miller RL, Braun SB. Disposition of cocaine in pregnant sheep. l. Pharmacokinetics. Dev Pharmacol Ther 1991;16:123-9. 19. Spear LP. Frambes NA, Kirstein CL. Fetal and maternal brain and plasma levels of cocaine and benzoylecgonine following chronic subcutaneous administration of cocaine during gestation in rats. Psychopharmacology (Berl) 1989; 97:427-31. 20. Schenker S. Yang Y, Johnson RF, et al. The transfer of cocaine and its metabolites across the term human placenta. Clin Pharmacol Ther 1993;53:329<)9. 21. Derewlany LO, Leeder .lS, Kumar R, Radde Ie. Knie B. Koren G. Transfer of digoxin across the perfused human placental lobule. J Pharmacol Exp Ther 1991 ;256: 11 0711. 22. Klein .I, Greenwald M, Becker L, Koren G. Fetal distribution of cocaine: a case analysis. Pediatr Pathol 1992; 12: 463-8. 23. Cone E.l, Mitchell J. Validity testing of commercial urme cocaine metabolite asqys. II. Sensitivity. specificity. accu-

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24. 25. 26. 27. 28.

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racy and confirmation by gas chromatography/mass spectrometry. j Forensic Sci 1989;34:32-45. Mittleman RE, Cofino jC, Hearn WL. Tissue distribution of cocaine in a pregnant woman. j Forensic Sci 1989;34: 481-6. Fox H, Faulk W. The placenta as an experimental animal. Clin Endocrinol Metab 1981;10:57-72. Fox H. Placenta as a model for organ aging. In: Beaconsfield P, Villee G, eds. Placenta-a neglected experimental animal. Oxford: Pergamon Press, 1979:351-85. Hytten F, Chamberlain G. Clinical physiology in obstetrics. Oxford: Blackwell Scientific Publishers, 1980. Ng W, Miller RK. Transport of nutrients in the early

human placenta: amino acid, creatinine, vitamin B 12 • Trophoblast Res 1983;1:121-34. 29. Dancis j, Levitz M, Katz D, et al. Transfer and metabolism of retinol by the perfused human placenta. Pediatr Res 1992;32:195-9. 30. Fortunato Sj, Bawdon RE, Swan KF, Bryant EC, Sobhi S. Transfer of Timentin (ticarcillin and clavulanic acid) across the in vitro perfused human placenta: comparison with other agents. AM j OBSTET GYNECOL 1992;167:159599. 31. Ahmed MS, Zhou DE, Maulik D, Eldefrawi ME. Characterization of a cocaine binding protein in human placenta. Life Sci 1990;46:553-61.

Differential c-jun gene expression with tonically administered steroids in rat ovary and uterus Martha E. Shelley, MD, Amjad Hossain, PhD, Paul G. McDonough, MD, and Iqbal Khan, PhD

Augusta, Georgia OBJECTIVE: The purpose of this study was to evaluate the induction of the early regulatory gene c-jun in response to tonic exposure to estradiol and progesterone in rat ovary, uterus, and adrenal tissues. STUDY DESIGN: Pellets containing estradiol-17j3, progesterone, and estradiol-17j3 plus progesterone were placed subcutaneously in immature female Sprague-Dawley rats (N = 24). The ovary, uterus, and the adrenal were evaluated for c-jun expression by Northern analysis at 24 and 48 hours. RESULTS: The c-jun messenger ribonucleic acid expression in the ovary and adrenal gland was inhibited with high, non physiologic doses of estradiol in progesterone and was induced with physiologic levels of estradiol. Physiologic levels of progesterone do not appear to influence the expression of c-jun in the ovary or adrenal gland. Uterine c-jun expression to estradiol and progesterone is generally the opposite of that observed in the ovary. CONCLUSION: These findings suggest that there is both tissue and dose specificity of c-jun gene expression in steroidogenic and steroid-responsive tissues when steroid hormones are tonically administered. (AM J OSSTET GVNECOL 1994;170:1410-5.)

Key words: Protooncogene, cjun, estradiol, progesterone The interplay of the two steroid hormones estrogen and progesterone and the growth and differentiation of different tissues in the reproductive system have been the topic of investigation over a long period of time. The physiologic role of steroids in the reproductive process is now fairly well described. I, 2 However, the molecular mechanisms by which steroid hormones and From the Department oj Obstetrics and Gynecology, Medical College of Georgza. Recezved for publicatzon June 8, 1993; reVised August 11, 1993; accepted November 12, 1993. Reprint requests: Iqbal Khan, PhD, Reproductive Endocrinology Sectzon, Cj-134, Department of ObstetriCs and Gynecology, Medlcal College of Georgza, Augusta, GA 30912. Copyright © 1994 by Mosby-Year Book, Inc.

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receptors interact to regulate genes participating in the growth or differentiation of reproductive tissues is currently under scrutiny.'·6 Recently the nuclear protooncogenes jun and fos, both components of the AP-l transcription factor, have been demonstrated to play a role in cell proliferation and differentiation and are good candidates for mediators of steroid-induced growth and differentiation. 7 '9 Some studies implicate c-jun as an "early response gene" in the cascade of steroid action on the target tissue. 10, II To date, the response of these protooncogenes to steroid hormones has been evaluated only at the physiologic level of these hormones and at short exposure times. However, in some situations the biologic system may be loaded with steroids for protracted periods far in excess of physio-