- Email: [email protected]
6 mouse brain relative to maternal brain and plasma
Neurotoxicology
ELSEVIER
and Teratology, Vol. 18, No. 6, pp. 645-649,1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0X92-0362/96 $15.00 + .OO
PI1SOS92-0362(96)00076-l
Cocaine Concentrations in Fetal C57BL/6 Mouse Brain Relative to Maternal Brain and Plasma SCOTT
R. MILLER*,
LAWRENCE D. MIDDAUGH,? WILLIAM KENNERLY S. PATRICK*
0. BOGCANt
AND
*Department of Pharmaceutical Sciences and TDepartment of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, SC 29425-0742
Received 16 August 199.5;Accepted 19 February 1996 MILLER. S. R., L. D. MIDDAUGH, W. 0. BOGGAN AND K. S. PATRICK. Cocaine concentrations in fetalC57BW6 mouse brain relative to maternal bruin and plasma. NEUROTOXICOL TERATOL 18(6) 645-649,1996.-Cocaine concentrations in maternal plasma and brain and fetal brain of mice were evaluated as a model for fetal brain exposure during maternal cocaine use. On days 12-18 of gestation, mice (CS7BLi6; N = 5-7/group) received SC cocaine-HCI: 20 or 40 mg/kg. Maternal plasma and brain (accumbens and caudate nuclei removed), and fetal brain were collected at 0.5, 1, and 2 h following the last injection. Analysis was by GC-MS. Brain cocaine levels in the dams declined from 9.6 to 3.4 and 20.9 to 12.5 mg/g during the 0.5-1-h period after the low and high doses, respectively, and were 7.5-14.3 times greater than plasma levels. The corresponding fetal brain concentrations changed from 1.6 to 1.3 and 2.9 to 3.4 mg/g. By 2 h, brain cocaine concentrations in dams declined to approximately 10% of their 0.5-h values, with a slower drug decay occurring in fetal brain. Maternal plasma cocaine concentrations correlated with those of maternal brain (r = 0.94, p < 0.01) and fetal brain (r = 0.69. p < 0.01). The present results indicate that cocaine accumulates to a lesser extent in fetal brain than in maternal brain of C57BL/6 mice; however, Ihe duration of exposure appears to be more sustained in the fetus, a phenomenon that may have toxicological implications for human in utero cocaine exposure. Copyright 0 1996 Elsrvier Science Inc. Cocaine
CS7BLi6
mouse
Brain
Plasma
Pregnancy
NEONATES of women who have used cocaine during pregnancy have presented with behavioral disorders (3) and submedian head circumference (1,3). Other cranial abnormalities have been demonstrated by ultrasonography and include subarachnoid hemorrhage, white matter cavities, and ventricular enlargement (6)-pathology related to that reported for adult cocaine abusers (15). However, the concurrent use of other drugs (e.g., alcohol, nicotine) has confounded a specific causal relationship [see (12) for commentary]. In animal models, cocaine has been directly implicated in fetal central nervous system toxicity; the underlying mechanisms may involve hypoxic tissue damage (16). disruption of neuronal differentiation (37), and/or alteration in the development of monoamine neurotransmitter systems (7,8,10,21,32).
Fetus
Establishing fetal and maternal brain cocaine concentrations in animal studies of developmental neurotoxicity may reveal meaningful pharmacodynamic and interstudy relationships, as well as offering a potential for extrapolation to human investigations. Although fetal brain cocaine concentrations associated with those of maternal cocaine have been reported in studies using rats (5,9,31), fetal and maternal brain cocaine concentrations in the mouse species is limited to a single radiotracer study of Swiss-Webster mice (29). dosed at 10 mg/kg IP and sampled at 0.25 and 3 h. In view of reported strain differences in brain cocaine disposition (34), and as an integral part of a research program to establish the long-term effects of prenatal cocaine exposure, the present study extends such cocaine distributional relation-
Requests for reprints should be addressed to Kennerly S. Patrick, Medical University of South Carolina, Department ences, 171 Ashley Avenue, Charleston. SC 29425.2303. Tel: (803) 792-8429; Fax: (803) 792.0759, E-mail: [email protected]
645
of Pharmaceutical
Sci-
MILLER
646
ships to maternal and fetal C57BL/6 mice. In contrast to the previous report on Swiss-Webster mice, in the present investigation cocaine was administered SC for multiple days and animals were sacrificed at 0.5, 1, and 2 h, a protocol more comparable to pregnant rat studies (5,9,31). The SC rather than IP route of administration has been commonly used in prenatal drug studies to avoid possible direct effects of cocaine on the uterine horn, to prevent potential physical injury to the fetus, and to assure the delivery of the drug to the fetus only via placental transfer from the general circulation. A modification of a recently developed gas chromatographic-mass spectrometric (GC-MS) analytical method (23) offered the requisite sensitivity and specificity for the microsample determinations involved. This report constitutes the first application of chromatographic methodology to cocaine levels in brains of fetal mice.
ET AL.
sample, followed by homogenization (Polytron) for 20 s and centrifugation (3000 X g) for 20 min. The clear supernatants were transferred to screw-cap culture tubes (13 X 100 mm), saturated aqueous sodium borate (1.5 ml) was added, and then these were extracted according to the method used for alkalinized plasma samples. Analyses utilized a Finnigan 4000 GC-MS, making splitless injections of acetonitrile-reconstituted samples onto a (5% phenyl)methylpolysiloxane fused-silica column, 30 m X 0.32 mm i.d., 0.25 p_rn film thickness (DB-Sms, J & W Scientific, Folsom. CA), maintained at 240°C with the helium carrier gas linear velocity at 55 cm/s. Detection was by selected ion monitoring of electron impact (70 eV) generated fragment ions of cocaine, m/z 182. and [‘H,]cocaine, m/z 185. Concentrations were calculated from the slope and intercept of the associated standard curve, plotted as peak area ratio (cocaine/[*H,]cocaine) vs. calibrator concentrations, and reported as the free base.
METHOD
Cocaine-HCl (batch #6907-1022-167 C, purity > 95%) and [2H,]cocaine-HCl (batch #f3995-6-B, purity: 99.77% [*H& 0.23% [2H2]) were obtained from the National Institute of Drug Abuse (Rockville, MD). Female C57BLi6 mice were purchased from Jackson Laboratories at 49 days of age. After a 2-week acclimatization period, the mice were bred according to an established procedure (17). The presence of a vaginal sperm plug after 12 h with a male defined the beginning of pregnancy, gestation day (G) 0. The mice were weighed and caged individually. On Gil pregnancy was confirmed by weight gain and the animals were assigned to drug treatment conditions. From G12 to G18, cocaine-HCl (20 or 40 mgikg) was injected SC [O.Ol ml/g body weight (30-41 g)] in the suprascapular region between 0830 and 1000 h. Blood was collected from the infraorbital sinus into 75-~1 capillary tubes and the animals decapitated 0.5, 1, and 2 h following the last dose (N = 5-7/group). The blood was immediately transferred to test tubes containing NaF (calculated to provide 0.3% w/v), maintained on ice until the end of the sacrifice session, and then spun for plasma. Brains were removed from the dam and fetuses within 3-5 min of sacrifice and all samples were frozen at -70°C until analysis by adaptation of an established GCMS method (23). For plasma analysis, aqueous sodium fluoride [l% (w/v), 0.25 ml] containing 100 ng of [*H?]cocaine-HCl was added to each sample (50 kl), followed by alkalization with saturated aqueous sodium borate (0.75 ml), extraction with 3 ml of pentaneiisopropanol (97:3), and evaporation to dryness under nitrogen. Calibration standards were extracted in parallel with the unknowns, used 50 ~1 of horse serum from Sigma Chemical (found to be substitutable for mouse plasma), and were spiked with cocaine-HCl in methanol (25 ng/kl) to provide concentrations of 0,0.2,0.5, 1,2,3,4, and 6 p,g/ml. Cocaine concentrations in brain tissue were determined from either two whole fetal brains (0.11-0.16 g) obtained from the same litter or from the dam’s brain (0.15-0.26 g) minus the accumbens and caudate nuclei, which were dissected out for neurochemical studies (to be reported elsewhere). Brain samples were placed in IS-ml Nalgene centrifuge tubes containing aqueous NaF [l% (w/v), 250 $1. Calibration standards, run in parallel with the unknowns, incorporated portions of blank mouse brain that were fortified with cocaine-HCl in methanol (200 ngipl) to typically provide 0,2.5,5, 10, 15,20, and 25 pg/g brain cocaine concentrations for the dam or 0, 1, 2, 4, 8, 10. and 12 pgig for the fetal samples. Perchloric acid (0.1 M, 1 ml) containing 1 pg of [*H,]cocaine-HCl was then added to each
RESLJLTS
As reported previously for adult male mouse plasma and brain cocaine determinations (23), the GC-MS method used for analysis of the dam and fetal brain microsamples in the present study provided sensitive detection and generated ion chromatograms free of interferences (Fig. 1). The relatively short retention time for cocaine (2.05 min) allowed a practical chromatographic throughput of 4 min per sample. The associated calibration plots (N = 9) demonstrated linear analyte response (mean r > 0.998). Mean cocaine concentrations in brain tissue from the dams declined from 9.6 to 3.4 and 20.9 to 12.5 kg/g during the 0.5-l-h period following the 20 and 40 mg/kg doses, respectively (Fig. 2). The corresponding fetal brain concentrations changed from 1.6 to 1.3 and 2.9 to 3.4 kg/g. By 2 h, the mean brain concentrations in dams given either dose declined to approximately 10% of the 0.5-h values whereas the 2-h fetal brain concentrations declined to 19% of the 0.5-h value for the low-dose animals and to 28% for the high-dose group. The cocaine concentrations determined for the 20 mg/kg dosing group, when compared to the 40 mgikg group, generally reflected the difference in the magnitude of the two doses.
m/z 182
5
m/z 185
0
I \,
z 20 0 10
!I\ / \, i
I, 1.6
TIME FIG.
1. Selected
ion
[*H3]cocaine (lower).
;,
,, 1.8
AFTER
j !.i ,,,, 1 2.0
/, 2.2
INJECTION
chromatograms
,I, - I 2.4
(min)
of cocaine
(upper)
and
The analyzed sample was derived from 0.12 g of fetal brain tissue and determined to contain 0.20 bgig cocaine. Vertical lines flanking the chromatographic peaks indicate the boundaries used for peak area integrations.
COCAINE
3.6 3.2 T
IN FETAL MATERNAL
AND MATERNAL
MOUSE
647
PLASMA .
MATERNAL
BRAIN
27.0 7
-5
0
5
10
MATERNAL
15
BRAIN
20
25
30
35
(pg/mg)
FIG. 3. Correlation of cocaine concentrations in maternal plasma and brain (r = 0.94, p < 0.01). [Brain samples did not include accumbens and caudate nuclei (see the Method section).]
FETAL
BRAIN
0.5
INTERVAL
BETWEEN
1 .o INJECTION
2.0 AND SACRIFICE
(hr)
FIG. 2. Brain and plasma cocaine concentrations (* SE). Pregnant C57BLi6 mice (N = 5-7igroup) were dosed from gestation day 12-18 with 20 mg/kg (0) or 40 mg/kg (m) cocaine-HCI SC. then brains and plasma were collected after the last dose. [Brain samples did not include accumbens and caudate nuclei (see the Method section).]
Fetal brain cocaine levels were 17% and 14% that of maternal brain levels at the 30-min time point for the respective low/high-dose groups. At 1 h after cocaine administration the corresponding values were 38% and 27%. Plasma and brain concentrations of cocaine were highly correlated in the dam (Fig. 3, r = 0.94, p < 0.01); however, mean brain levels ranged from 7.5 to 14.3 times greater than those of plasma. Maternal plasma cocaine concentrations correlated to a lesser extent with the fetal brain levels (Fig. 4, r = 0.69, p < 0.01) and fetal brain/dam plasma ratios ranged from 1.0 to 4.4.
esterase activity (11) and amniotic fluid serving as a drug reservoir (28) may all be pertinent to this comparatively slow decay of cocaine from fetal brain. Cocaine concentrations in the 2-h plasma and brain samples from the C57BL/6 mice were considerably lower than the corresponding 0.5-h concentrations (Fig. 2). This concentration-time relationship was not found in a related study using pregnant Sprague-Dawley rats and trunk blood collection (31). Though successive-day 20 and 40 mg/kg SC cocaine doses were administrated in both the mouse and rat studies (injection drug concentrations differed), in the rats the 2-h maternal plasma and brain cocaine levels, as well as those of fetal brain, were elevated rather than reduced when compared to the 0.5-h concentrations. This inconsistency between studies may be related to species differences (e.g., skin thickness and fat distribution altering SC cocaine absorption rates). Further, note that even strain differences among mice have been reported to influence cocaine disposition [administered IP (34)]. The longer duration of dosing used in the rat protocol (G8-20) than in the mouse protocol (G12-18) may also have contributed to interstudy drug level disparities; indeed, chronicity has been reported to significantly increase cocaine bioavailability (22). However, conclusions regarding pharmacokinetic differences between these two animal mod-
1
DISCUSSION
The moderate accumulation of cocaine in fetal C57BW6 mouse brain (Fig. 2) is in general agreement with the OS- and l-h fetal brain/maternal plasma cocaine concentration ratios reported for Wistar rats [maternal rat brain was not analyzed (9)J The lower correlation of the dam’s plasma cocaine concentrations with fetal brain (Fig. 4) than with her own brain cocaine values (Fig. 3) reflects. at least in part, the retarded decline of cocaine from fetal brain when compared to maternal plasma. Drug redistribution, lower fetal pH, lower fetal
0
1
2
FETAL
3
BRAIN
4
5
(pg/g)
FIG. 4. Correlation of cocaine concentrations and fetal brain (r = 0.69, p < 0.01).
in maternal plasma
MILLER
648 els are limited by the absence of l-h cocaine concentrations in the rat study. In the present study, blood samples were collected from the infraorbital sinus rather than as trunk blood to avoid possible overestimation of cocaine, which could result from blood mixing with stomach contents during trunk blood harvesting. Such an overestimation may occur even after parenterally administering cocaine because the drug potentially attains high concentrations in the stomach relative to plasma through the ion trapping effect common to basic drugs (26,30,33). This concern is heightened by the report of a threefold greater area under the cocaine concentration-time curve for trunk blood when compared to tail blood concentrations in Holtzman rats (14). However, it is recognized that large differences in arteriovenous drug concentrations may exist, whereby blood cocaine concentrations become highly dependent upon the specific sampling site (4) beyond any stomach content considerations. The pronounced accumulation of cocaine in maternal CS7BU6 mouse brain (Fig. 2) is a distributional characteristic of other lipophilic drugs (24). Although little (28,31) or no (5) accumulation of cocaine in adult brain has been reported for guinea pigs or Sprague-Dawley rats, the high brain/plasma cocaine concentration ratios determined in the C57BLi6 mice of the present study are in good agreement with findings using male (25) or female (2) BALB/cBy mice, maternal SwissWebster mice (29) male ICR mice (27) male Wistar rats (l&20), or maternal Long-Evans rats (35). Because the accumbens and caudate were not included in the maternal brain samples analyzed in the present study (see the Method section), and because regional differences in cocaine concentrations within brain have been reported [in rats (13)], the partial
ET AL.
brain concentrations shown in Fig. 2 may be at some variance with whole brain values. Had the excised brain regions been included in the analyses, even higher brain/plasma cocaine concentration ratios would be expected because most of the weight of the removed tissue was caudate, a brain region associated with high relative cocaine accumulation (13). The findings that cocaine accumulated in fetal C57BLi6 mouse brain to a lesser extent than in maternal brain (Fig. 2) are consistent with cocaine dispositional studies using SwissWebster mice (29) or Long-Evans rats (35). Further, in fetal Sprague-Dawley rat brain cocaine concentrations were generally less than or equal to maternal plasma levels (31). This relatively low, or lack of, cocaine accumulation in fetal brain is potentially attributable to the lower lipid content in fetal brain than in maternal brain (11,35) and/or to fetal drug availability being compromised by a cocaine induced reduction in uterine blood flow (19,36). In summary, although the results of this investigation indicate that cocaine accumulates to a lesser extent in fetal brain than in maternal brain of C57BL/6 mice, the duration of exposure appears to be more sustained in the fetus (Fig. 2) a phenomenon that may have toxicological implications for human in utero cocaine exposure. ACKNOWLEDGEMENTS
This research was supported by the National Institute of Drug Abuse (DA08034) to L. D. Middaugh. These results were presented at the 23rd Annual Meeting of the American College of Clinical Pharmacology (abstract in J. Clin. Pharmacol. 43:1024; 1994). The authors wish to thank Ms. Paula Bishop for her help in preparing this manuscript and Ms. R. Ruotolo for assistance with data analysis and graphics.
REFERENCES I. Bateman, D. A.; Ng, S. K. C.; Hansen, C. A.; Heagarty, M. C. The effects of intrauterine cocaine exposure in newborns. Am. J. Public Health 83:190-193; 1993. 2. Benuck, M.; Lajtha, A.: Reith, M. E. A. Pharmacokinetics of systemically administered cocaine and locomotor stimulation in mice. J. Pharmacol. Exp. Ther. 243:1444149; 1987. 3. Chasnoff, I. J.; Lewis, D. E.; Griffith, D. R.; Willey. J. Cocaine and pregnancy: clinical and toxicological implications for the neonate. Clin. Chem. 35:1276-1278; 1989. and rationale of marked depen4. Chiou, W. L. The phenomenon dence of drug concentration on blood sampling site: Implications in pharmacokinetics, pharmacodynamics, toxicology and therapeutics. Clin. Pharmacokinet. 17:175-199; 1989. .5 Devane. C. L.; Simpkins, J. W.; Miller, R. L.; Braun. S. B. Tissue distribution of cocaine in the pregnant rat. Life Sci. 45:1271-1276; 1989. findings in neo6. Dixon, S. D.: Bejar. R. Echoencephalographic nates associated with maternal cocaine and methamphetamine use: incidence and clinical correlates. J. Pediatr. 115:77@778; 1989. D. L.; Freed, L. A.; Milhorat, T. H. Simulation of 7. Dow-Edwards, brain metabolism by perinatal cocaine exposure. Dev. Brain Res. 42:137-141; 1988. D. L. Developmental effects of cocaine, NIDA 8. Dow-Edwards, Res. Monogr., 88:29@303: 1988. D. Fetal and maternal cocaine levels peak rapidly Y. Dow-Edwards, following intragastric administration in the rat. J. Subst. Abuse 21427-437; 1990. D. L.; Freed. L. A.: Fico, J. A. Structural and 10. Dow-Edwards, functional effects of prenatal cocaine exposure in adult rat brain. Dev. Brain Res. S7:263-268; 1990. 1I. Finnegen, L. P.; Mellott. J. M.; Williams, L. R.; Wapner, R. J. Perinatal exposure to cocaine: human studies. In: Lakoski, J. M.; Galloway, M. P.; White. F. J., eds. Cocaine pharmacology, physi-
12.
13. 14.
15.
16.
17.
18.
19.
20.
21.
ology, and clinical strategies. Boca Raton, FL: CRC Press; 1992:391409. Hutchings, D. E. The puzzle of cocaine’s effects following maternal use during pregnancy: Are there reconcilable differences? Neurotoxicol. Teratol. 15:281-286; 1993. Javaid, J. I.: Davis, J. M. Cocaine disposition in discrete regions of rat brain. Biopharm. Drug Dispos. 14:357-364; 1993. Lau, C. E.; Iman, A.; Ma, F.; Falk. J. L. Acute effects of cocaine on spontaneous and discriminative motor functions: Relation to route of administration and pharmacokinetics. J. Pharmacol. Exp. Ther. 2.571444456; 1991. Levine, S. R.; Washington, J. M.; Jefferson, M. F.; Kieran, S. N.; Moen, M.; Feit, H.; Welsh, K. M. A. “Crack” cocaine-associated stroke. Neurology 37:1849-1853; 1987. MahaIik, M. P.; Gautieri, R. F.; Mann, D. E., Jr. Mechanisms of cocaine-induced teratogenesis. Res. Commun. Subst. Abuse 5:279302; 1984. Middaugh, L. D.; Boggan, W. 0. Postnatal growth deficits in prenatal ethanol exposed mice: Characteristics and critical periods. Alcohol. Clin. Exp. Res. 15:919-926; 1991. Misra, A. L.; Nayak, P. K.; Patel, M. N.; Vadlamani, M. L.; Mule, S. J. Identification of norcocaine as a metabolite of [“HI-cocaine in rat brain. Experientia 30:1312-1314; 1974. Morishima, H. 0.; Cooper, T. B.; Hara, T.; Miller, E. D., Jr. Pregnancy alters the hemodynamic responses to cocaine in the rat. Dev. Pharmacol. Ther. 19:69-79; 1992. Nayak, P. K.; Misra, A. L.; Mule, S. J. Physiological disposition and biotransformation of [3H]cocaine in acutely and chronically treated rats. J. Pharmacol. Exp. Ther. 396:556-569; 1976. Needleman, R.; Zuckerman, B.; Anderson, G. M.; Mirochnick, M.; Cohen, D. J. Cerebrospinal fluid monoamine precursors and metabolites in human neonates following in utero cocaine exposure: A preliminary study. Pediatrics 92:55-60; 1993.
COCAINE
IN
FETAL
AND
MATERNAL
MOUSE
22. Pan. H. T.; Menacherry, S.: Justice, Jr., J. B.. Differences in the pharmacokinetics of cocaine in naive and cocaine-experienced rats. J. Neurochem. 56:1299-1306; 1991. 23. Patrick, K. S.; Boggan, W. 0.; Miller, S. R.; Middaugh, L. D. Gas chromatographic-mass spectrometric determination of plasma and brain cocaine in mice. J. Chromatogr. B Biomed. Appl. 621:X9-94; 1993. 24. Patrick, K. S.; Ellington, K. R.; Breese, G. R. Distribution of methylphenidate and p-hydroxymethylphenidate in rats. J. Pharmacol. Exp. Ther. 231:61-65; 1984. 2s. Reith, M. E. A.: Benuck, M.; Lajtha, A. Cocaine disposition in the brain after continuous or intermittent treatment and locomotor stimulation in mice. J. Pharmacol. Exp. Ther. 243:281-2X7: 1987. 26. Ritscef. W. A.; Adolph, S.; Denson. D. D. lon-trapping of meperidine: Influence of antacid treatments on serum and gastric fluid concentrations. Methods Find. Exp. Clin. Pharmacol. 12:47-51; 1990. 27. Roberts, S. M.; Phillips, D. L.: Tebbett, 1. R. Increased blood and brain cocaine concentrations with ethanol cotreatment in mice. Drug Metab. Disp. 23:664-666; 1995. 28. Sandberg, J. A.; Olsen, G. D. Cocaine and metabolite concentrations in the fetal guinea pig after chronic maternal cocaine administration. J. Pharmacol. Exp. Ther. 2601587-591; 1991. 29. Shah, N. S.: May. D. A.; Yates, J. D. Disposition of levo[“HIcocaine in pregnant and nonpregnant mice. Toxicol. Appl. Pharmacol. 53:2792X4: 1980.
649
30. Shore, P. A.; Brodie. B. B.; Hogben, C. A. M. The gastric secretion of drugs: A pH partition hypothesis. J. Pharmacol. Exp. Ther. 119:361-369; 1956. 31. Spear, L. P.; Frambes, N. A.; Kirstein. C. L. Fetal and maternal brain and plasma levels of cocaine and benzoylecgonine following chronic SC administration of cocaine during gestation in rats. Psychopharmacology (Berlin) 97:427431; 1989. 32. Spear, L. P.; Kirstein, C. L.; Frambes, N. A. Cocaine effects on the developing CNS: Behavioral, psychopharmacological and neurochemical studies. Ann. NY Acad. Sci. 562:290-307; 1989. 33. Stoeckel, H.; Hengstmann, J. H.; Schuttler, J. Pharmacokinetics of fentanyl as a possible explanation for recurrence of respiratory depression. Br. J. Anaesth. 51:741-S: 1979. 34. Weiner. L. H.; Reith, M. E. A. Correlation between cocaineinduced locomotion and cocaine disposition in the brain among four inbred strains of mice. Pharmacol. Biochem. Behav. 36:699701 ; 1990. 35. Wiggins, R. C.: Rolsten. C.; Rutz. 8.; Davis, C. M. Pharmacokinetics of cocaine: Basic studies of route, dosage, pregnancy and lactation. Neurotoxicology 10:367-382; 1989. 36. Woods. J. R.: Plessinger, M. A.: Clark. K. E. Effect of cocaine on uterine blood flow and fetal oxygenation. J. Am. Med. Assoc. 257:957-961: 1987. 37. Zachor, D.: Cherkes, J. K.; Fay. C. T.; Ocrant. 1. Cocaine differentially inhibits neuronal differentiation and proliferation in vivo. J. Clin. Invest. 93: 1179-l 185; 1994.
ELSEVIER
and Teratology, Vol. 18, No. 6, pp. 645-649,1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0X92-0362/96 $15.00 + .OO
PI1SOS92-0362(96)00076-l
Cocaine Concentrations in Fetal C57BL/6 Mouse Brain Relative to Maternal Brain and Plasma SCOTT
R. MILLER*,
LAWRENCE D. MIDDAUGH,? WILLIAM KENNERLY S. PATRICK*
0. BOGCANt
AND
*Department of Pharmaceutical Sciences and TDepartment of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, SC 29425-0742
Received 16 August 199.5;Accepted 19 February 1996 MILLER. S. R., L. D. MIDDAUGH, W. 0. BOGGAN AND K. S. PATRICK. Cocaine concentrations in fetalC57BW6 mouse brain relative to maternal bruin and plasma. NEUROTOXICOL TERATOL 18(6) 645-649,1996.-Cocaine concentrations in maternal plasma and brain and fetal brain of mice were evaluated as a model for fetal brain exposure during maternal cocaine use. On days 12-18 of gestation, mice (CS7BLi6; N = 5-7/group) received SC cocaine-HCI: 20 or 40 mg/kg. Maternal plasma and brain (accumbens and caudate nuclei removed), and fetal brain were collected at 0.5, 1, and 2 h following the last injection. Analysis was by GC-MS. Brain cocaine levels in the dams declined from 9.6 to 3.4 and 20.9 to 12.5 mg/g during the 0.5-1-h period after the low and high doses, respectively, and were 7.5-14.3 times greater than plasma levels. The corresponding fetal brain concentrations changed from 1.6 to 1.3 and 2.9 to 3.4 mg/g. By 2 h, brain cocaine concentrations in dams declined to approximately 10% of their 0.5-h values, with a slower drug decay occurring in fetal brain. Maternal plasma cocaine concentrations correlated with those of maternal brain (r = 0.94, p < 0.01) and fetal brain (r = 0.69. p < 0.01). The present results indicate that cocaine accumulates to a lesser extent in fetal brain than in maternal brain of C57BL/6 mice; however, Ihe duration of exposure appears to be more sustained in the fetus, a phenomenon that may have toxicological implications for human in utero cocaine exposure. Copyright 0 1996 Elsrvier Science Inc. Cocaine
CS7BLi6
mouse
Brain
Plasma
Pregnancy
NEONATES of women who have used cocaine during pregnancy have presented with behavioral disorders (3) and submedian head circumference (1,3). Other cranial abnormalities have been demonstrated by ultrasonography and include subarachnoid hemorrhage, white matter cavities, and ventricular enlargement (6)-pathology related to that reported for adult cocaine abusers (15). However, the concurrent use of other drugs (e.g., alcohol, nicotine) has confounded a specific causal relationship [see (12) for commentary]. In animal models, cocaine has been directly implicated in fetal central nervous system toxicity; the underlying mechanisms may involve hypoxic tissue damage (16). disruption of neuronal differentiation (37), and/or alteration in the development of monoamine neurotransmitter systems (7,8,10,21,32).
Fetus
Establishing fetal and maternal brain cocaine concentrations in animal studies of developmental neurotoxicity may reveal meaningful pharmacodynamic and interstudy relationships, as well as offering a potential for extrapolation to human investigations. Although fetal brain cocaine concentrations associated with those of maternal cocaine have been reported in studies using rats (5,9,31), fetal and maternal brain cocaine concentrations in the mouse species is limited to a single radiotracer study of Swiss-Webster mice (29). dosed at 10 mg/kg IP and sampled at 0.25 and 3 h. In view of reported strain differences in brain cocaine disposition (34), and as an integral part of a research program to establish the long-term effects of prenatal cocaine exposure, the present study extends such cocaine distributional relation-
Requests for reprints should be addressed to Kennerly S. Patrick, Medical University of South Carolina, Department ences, 171 Ashley Avenue, Charleston. SC 29425.2303. Tel: (803) 792-8429; Fax: (803) 792.0759, E-mail: [email protected]
645
of Pharmaceutical
Sci-
MILLER
646
ships to maternal and fetal C57BL/6 mice. In contrast to the previous report on Swiss-Webster mice, in the present investigation cocaine was administered SC for multiple days and animals were sacrificed at 0.5, 1, and 2 h, a protocol more comparable to pregnant rat studies (5,9,31). The SC rather than IP route of administration has been commonly used in prenatal drug studies to avoid possible direct effects of cocaine on the uterine horn, to prevent potential physical injury to the fetus, and to assure the delivery of the drug to the fetus only via placental transfer from the general circulation. A modification of a recently developed gas chromatographic-mass spectrometric (GC-MS) analytical method (23) offered the requisite sensitivity and specificity for the microsample determinations involved. This report constitutes the first application of chromatographic methodology to cocaine levels in brains of fetal mice.
ET AL.
sample, followed by homogenization (Polytron) for 20 s and centrifugation (3000 X g) for 20 min. The clear supernatants were transferred to screw-cap culture tubes (13 X 100 mm), saturated aqueous sodium borate (1.5 ml) was added, and then these were extracted according to the method used for alkalinized plasma samples. Analyses utilized a Finnigan 4000 GC-MS, making splitless injections of acetonitrile-reconstituted samples onto a (5% phenyl)methylpolysiloxane fused-silica column, 30 m X 0.32 mm i.d., 0.25 p_rn film thickness (DB-Sms, J & W Scientific, Folsom. CA), maintained at 240°C with the helium carrier gas linear velocity at 55 cm/s. Detection was by selected ion monitoring of electron impact (70 eV) generated fragment ions of cocaine, m/z 182. and [‘H,]cocaine, m/z 185. Concentrations were calculated from the slope and intercept of the associated standard curve, plotted as peak area ratio (cocaine/[*H,]cocaine) vs. calibrator concentrations, and reported as the free base.
METHOD
Cocaine-HCl (batch #6907-1022-167 C, purity > 95%) and [2H,]cocaine-HCl (batch #f3995-6-B, purity: 99.77% [*H& 0.23% [2H2]) were obtained from the National Institute of Drug Abuse (Rockville, MD). Female C57BLi6 mice were purchased from Jackson Laboratories at 49 days of age. After a 2-week acclimatization period, the mice were bred according to an established procedure (17). The presence of a vaginal sperm plug after 12 h with a male defined the beginning of pregnancy, gestation day (G) 0. The mice were weighed and caged individually. On Gil pregnancy was confirmed by weight gain and the animals were assigned to drug treatment conditions. From G12 to G18, cocaine-HCl (20 or 40 mgikg) was injected SC [O.Ol ml/g body weight (30-41 g)] in the suprascapular region between 0830 and 1000 h. Blood was collected from the infraorbital sinus into 75-~1 capillary tubes and the animals decapitated 0.5, 1, and 2 h following the last dose (N = 5-7/group). The blood was immediately transferred to test tubes containing NaF (calculated to provide 0.3% w/v), maintained on ice until the end of the sacrifice session, and then spun for plasma. Brains were removed from the dam and fetuses within 3-5 min of sacrifice and all samples were frozen at -70°C until analysis by adaptation of an established GCMS method (23). For plasma analysis, aqueous sodium fluoride [l% (w/v), 0.25 ml] containing 100 ng of [*H?]cocaine-HCl was added to each sample (50 kl), followed by alkalization with saturated aqueous sodium borate (0.75 ml), extraction with 3 ml of pentaneiisopropanol (97:3), and evaporation to dryness under nitrogen. Calibration standards were extracted in parallel with the unknowns, used 50 ~1 of horse serum from Sigma Chemical (found to be substitutable for mouse plasma), and were spiked with cocaine-HCl in methanol (25 ng/kl) to provide concentrations of 0,0.2,0.5, 1,2,3,4, and 6 p,g/ml. Cocaine concentrations in brain tissue were determined from either two whole fetal brains (0.11-0.16 g) obtained from the same litter or from the dam’s brain (0.15-0.26 g) minus the accumbens and caudate nuclei, which were dissected out for neurochemical studies (to be reported elsewhere). Brain samples were placed in IS-ml Nalgene centrifuge tubes containing aqueous NaF [l% (w/v), 250 $1. Calibration standards, run in parallel with the unknowns, incorporated portions of blank mouse brain that were fortified with cocaine-HCl in methanol (200 ngipl) to typically provide 0,2.5,5, 10, 15,20, and 25 pg/g brain cocaine concentrations for the dam or 0, 1, 2, 4, 8, 10. and 12 pgig for the fetal samples. Perchloric acid (0.1 M, 1 ml) containing 1 pg of [*H,]cocaine-HCl was then added to each
RESLJLTS
As reported previously for adult male mouse plasma and brain cocaine determinations (23), the GC-MS method used for analysis of the dam and fetal brain microsamples in the present study provided sensitive detection and generated ion chromatograms free of interferences (Fig. 1). The relatively short retention time for cocaine (2.05 min) allowed a practical chromatographic throughput of 4 min per sample. The associated calibration plots (N = 9) demonstrated linear analyte response (mean r > 0.998). Mean cocaine concentrations in brain tissue from the dams declined from 9.6 to 3.4 and 20.9 to 12.5 kg/g during the 0.5-l-h period following the 20 and 40 mg/kg doses, respectively (Fig. 2). The corresponding fetal brain concentrations changed from 1.6 to 1.3 and 2.9 to 3.4 kg/g. By 2 h, the mean brain concentrations in dams given either dose declined to approximately 10% of the 0.5-h values whereas the 2-h fetal brain concentrations declined to 19% of the 0.5-h value for the low-dose animals and to 28% for the high-dose group. The cocaine concentrations determined for the 20 mg/kg dosing group, when compared to the 40 mgikg group, generally reflected the difference in the magnitude of the two doses.
m/z 182
5
m/z 185
0
I \,
z 20 0 10
!I\ / \, i
I, 1.6
TIME FIG.
1. Selected
ion
[*H3]cocaine (lower).
;,
,, 1.8
AFTER
j !.i ,,,, 1 2.0
/, 2.2
INJECTION
chromatograms
,I, - I 2.4
(min)
of cocaine
(upper)
and
The analyzed sample was derived from 0.12 g of fetal brain tissue and determined to contain 0.20 bgig cocaine. Vertical lines flanking the chromatographic peaks indicate the boundaries used for peak area integrations.
COCAINE
3.6 3.2 T
IN FETAL MATERNAL
AND MATERNAL
MOUSE
647
PLASMA .
MATERNAL
BRAIN
27.0 7
-5
0
5
10
MATERNAL
15
BRAIN
20
25
30
35
(pg/mg)
FIG. 3. Correlation of cocaine concentrations in maternal plasma and brain (r = 0.94, p < 0.01). [Brain samples did not include accumbens and caudate nuclei (see the Method section).]
FETAL
BRAIN
0.5
INTERVAL
BETWEEN
1 .o INJECTION
2.0 AND SACRIFICE
(hr)
FIG. 2. Brain and plasma cocaine concentrations (* SE). Pregnant C57BLi6 mice (N = 5-7igroup) were dosed from gestation day 12-18 with 20 mg/kg (0) or 40 mg/kg (m) cocaine-HCI SC. then brains and plasma were collected after the last dose. [Brain samples did not include accumbens and caudate nuclei (see the Method section).]
Fetal brain cocaine levels were 17% and 14% that of maternal brain levels at the 30-min time point for the respective low/high-dose groups. At 1 h after cocaine administration the corresponding values were 38% and 27%. Plasma and brain concentrations of cocaine were highly correlated in the dam (Fig. 3, r = 0.94, p < 0.01); however, mean brain levels ranged from 7.5 to 14.3 times greater than those of plasma. Maternal plasma cocaine concentrations correlated to a lesser extent with the fetal brain levels (Fig. 4, r = 0.69, p < 0.01) and fetal brain/dam plasma ratios ranged from 1.0 to 4.4.
esterase activity (11) and amniotic fluid serving as a drug reservoir (28) may all be pertinent to this comparatively slow decay of cocaine from fetal brain. Cocaine concentrations in the 2-h plasma and brain samples from the C57BL/6 mice were considerably lower than the corresponding 0.5-h concentrations (Fig. 2). This concentration-time relationship was not found in a related study using pregnant Sprague-Dawley rats and trunk blood collection (31). Though successive-day 20 and 40 mg/kg SC cocaine doses were administrated in both the mouse and rat studies (injection drug concentrations differed), in the rats the 2-h maternal plasma and brain cocaine levels, as well as those of fetal brain, were elevated rather than reduced when compared to the 0.5-h concentrations. This inconsistency between studies may be related to species differences (e.g., skin thickness and fat distribution altering SC cocaine absorption rates). Further, note that even strain differences among mice have been reported to influence cocaine disposition [administered IP (34)]. The longer duration of dosing used in the rat protocol (G8-20) than in the mouse protocol (G12-18) may also have contributed to interstudy drug level disparities; indeed, chronicity has been reported to significantly increase cocaine bioavailability (22). However, conclusions regarding pharmacokinetic differences between these two animal mod-
1
DISCUSSION
The moderate accumulation of cocaine in fetal C57BW6 mouse brain (Fig. 2) is in general agreement with the OS- and l-h fetal brain/maternal plasma cocaine concentration ratios reported for Wistar rats [maternal rat brain was not analyzed (9)J The lower correlation of the dam’s plasma cocaine concentrations with fetal brain (Fig. 4) than with her own brain cocaine values (Fig. 3) reflects. at least in part, the retarded decline of cocaine from fetal brain when compared to maternal plasma. Drug redistribution, lower fetal pH, lower fetal
0
1
2
FETAL
3
BRAIN
4
5
(pg/g)
FIG. 4. Correlation of cocaine concentrations and fetal brain (r = 0.69, p < 0.01).
in maternal plasma
MILLER
648 els are limited by the absence of l-h cocaine concentrations in the rat study. In the present study, blood samples were collected from the infraorbital sinus rather than as trunk blood to avoid possible overestimation of cocaine, which could result from blood mixing with stomach contents during trunk blood harvesting. Such an overestimation may occur even after parenterally administering cocaine because the drug potentially attains high concentrations in the stomach relative to plasma through the ion trapping effect common to basic drugs (26,30,33). This concern is heightened by the report of a threefold greater area under the cocaine concentration-time curve for trunk blood when compared to tail blood concentrations in Holtzman rats (14). However, it is recognized that large differences in arteriovenous drug concentrations may exist, whereby blood cocaine concentrations become highly dependent upon the specific sampling site (4) beyond any stomach content considerations. The pronounced accumulation of cocaine in maternal CS7BU6 mouse brain (Fig. 2) is a distributional characteristic of other lipophilic drugs (24). Although little (28,31) or no (5) accumulation of cocaine in adult brain has been reported for guinea pigs or Sprague-Dawley rats, the high brain/plasma cocaine concentration ratios determined in the C57BLi6 mice of the present study are in good agreement with findings using male (25) or female (2) BALB/cBy mice, maternal SwissWebster mice (29) male ICR mice (27) male Wistar rats (l&20), or maternal Long-Evans rats (35). Because the accumbens and caudate were not included in the maternal brain samples analyzed in the present study (see the Method section), and because regional differences in cocaine concentrations within brain have been reported [in rats (13)], the partial
ET AL.
brain concentrations shown in Fig. 2 may be at some variance with whole brain values. Had the excised brain regions been included in the analyses, even higher brain/plasma cocaine concentration ratios would be expected because most of the weight of the removed tissue was caudate, a brain region associated with high relative cocaine accumulation (13). The findings that cocaine accumulated in fetal C57BLi6 mouse brain to a lesser extent than in maternal brain (Fig. 2) are consistent with cocaine dispositional studies using SwissWebster mice (29) or Long-Evans rats (35). Further, in fetal Sprague-Dawley rat brain cocaine concentrations were generally less than or equal to maternal plasma levels (31). This relatively low, or lack of, cocaine accumulation in fetal brain is potentially attributable to the lower lipid content in fetal brain than in maternal brain (11,35) and/or to fetal drug availability being compromised by a cocaine induced reduction in uterine blood flow (19,36). In summary, although the results of this investigation indicate that cocaine accumulates to a lesser extent in fetal brain than in maternal brain of C57BL/6 mice, the duration of exposure appears to be more sustained in the fetus (Fig. 2) a phenomenon that may have toxicological implications for human in utero cocaine exposure. ACKNOWLEDGEMENTS
This research was supported by the National Institute of Drug Abuse (DA08034) to L. D. Middaugh. These results were presented at the 23rd Annual Meeting of the American College of Clinical Pharmacology (abstract in J. Clin. Pharmacol. 43:1024; 1994). The authors wish to thank Ms. Paula Bishop for her help in preparing this manuscript and Ms. R. Ruotolo for assistance with data analysis and graphics.
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