Fetal disposition of Δ9-tetrahydrocannabinol (THC) during late pregnancy in the rhesus monkey

315-321(1987)

TOXICOLOGYANDAPPLlEDPHARMACOLOGY~,

Fetal Disposition

of Ag-Tetrahydrocannabinol (THC) during Late Pregnancy in the Rhesus Monkey

J. R. BAILEY,* H. C. CUNNY,*,-~ M. G. PAULE,*,-~AND W. SLIKKER, JR.*,? *Division ofReproductive and Developmental Toxicology, National Centerfor Toxicological Research, Jefferson, Arkansas 72079, and TDepartment ofPharmacology and Interdisciplinary Toxicology, University ofArkansas for Medical Sciences, Little Rock, Arkansas 72205

Received

November

7, 1986; accepted

April

15, 1987

Fetal Disposition of A9-Tetrahydrocannabinol (THC) during Late Pregnancy in the Rhesus Monkey. BAILEY,J.R.,CUNNY,H.C.,PAULE,M.G.,ANDSLIKKER,W.,JR.(~~~~). Toxicol. Appl. Pharmacol. 90, 3 15-32 1. Three late-term (Gestational Days 146- 15 1) rhesus monkeys were given 0.3 mg/kg A’-tetrahydrocannabinol (THC) intravenously via the maternal radial vein to quantify the placental transfer and fetal disposition of THC, the major psychoactive component of marijuana. Simultaneous blood samples were obtained from a maternal uterine vein and an intraplacental artery at 0,3, 15,30,45,60,90, 120, and 180 min after dosing using an intraplacental cannulation technique. Samples of fetal plasma, spleen, testis, lung, brain, liver, bile, kidney, adrenals, thymus, and placenta were obtained at 180 min postdose. Samples were analyzed for THC and a major metabolite, 11-nor-9-carboxy-THC (1 l-nor), by radioimmunoassay (RIA). Peak plasma THC values were obtained 3 and 15 min after dosing in the mother ( I4 19 t&ml) and fetus (83 rig/ml), respectively. By 3 hr, maternal and fetal plasma THC levels were equal (37 rig/ml). Maternal plasma was sampled beyond 180 min at 24,48, 72, and 96 hr postdose, times at which THC and 1l-nor concentrations were either near or at the lower limit of sensitivity for the RIA (2 rig/ml). While maternal plasma 1l-nor levels peaked at 1 hr (44 r&ml), almost no 1 l-nor wasdetected in fetal plasma (~5 @ml). Fetal tissue concentrations of THC were 53 -t 6 rig/g (SE) for brain and 114 + 10 rig/g for liver, while 1 l-nor was undetectable in placenta, fetal liver, and fetal brain. These data demonstrate that THC rapidly crosses the placenta and enters the fetus. The lack of 1l-nor in fetal plasma and tissues suggests that this metabolite does not readily cross the placenta and that the fetus does not readily metabolize THC to 1l-nor at this stage of development. A portion of this data was presented at the 1985 meeting of The American Society of Primatologists and at the 1986 meeting of The Teratology Society. This work was supported in part by NIDA IAG 224-83-0005. o 1987 Academic FWSS, II-C.

The high level of marijuana use over the past 15 years has heightened concern for the potential toxic effects related to A’-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana (Mechoulam, 1970). Use of marijuana by pregnant women and women of child-bearing age raises additional concern about the potential for adverse effects of THC on the developing conceptus. Approximately 13% of the women polled in a recent study reported using marijuana during the course of their pregnancy (Fried et al., 1983). Studies using the rat, mouse, hamster, 315

and rabbit have provided information on the absorption, distribution, placental transfer, metabolism, and excretion of THC (Agurell et al., 1969, 1970; Christensen et al., 1971; Freudenthal et al., 1972; Harbison and Mantilla-Plata, 1972; Harvey, 1984; Ho et al., 1970; Persaud and Ellington, 1968; Joneja, 1976, 1977; Kennedy and Waddell, 1972; Klausner and Dingell, 197 1; Layman and Milton, 1971; Vardaris et al., 1976). However, controversy still exists over the rate of transfer of THC across the placenta. Some investigators have suggested that THC is trans0041-008X/87

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Copyright 0 1987 by Academic F’ress, Inc. All rights of reproduction in any form reserved.

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ferred across the placenta in a very limited manner with most of a given dose remaining within the maternal compartment (Pace et al., 197 1; Freudenthal et al., 1972; Kennedy and Waddell, 1972; Ryrfeldt et al., 1973). Conversely, research with the pregnant hamster has indicated that THC readily crosses the placenta and reaches concentrations in the fetus that are three times higher when given in the first trimester than when given in the third trimester (Evans and Harbison, 1977). Observations from these studies suggest species- and gestational age-dependent placental transport. Teratogenic effects have been reported in animal studies with marijuana extract, THC, and marijuana smoke (Abel, 1980; Harbison and Mantilla-Plata, 1972; Lemberger et al., 1972; Vardaris et al., 1976). These effects are dependent on dose level, route of administration, gestational age at exposure, and species. These studies suggest that early gestational exposure can induce embryo toxicity and that late gestational exposure can result in neurological defects. Extrapolation of these animal data to humans is difficult. To aid in the assessment of the possible risk of human THC exposure in utero, experimentation with nonhuman primates is warranted. The utility of the rhesus monkey to model placental transfer of THC in pregnant humans is based on the similarities of placental type and function in the pregnant human and monkey. Currently, no time-course plasma distribution data for THC are available for the pregnant monkey, the pregnant human, or the fetus of either species. There is however, a singie time point, postdelivery report of cannabinoid concentrations in human fetal cord blood and maternal blood (Blackard and Tennes, 1984). These samples were obtained from women who reported having smoked marijuana within 5-26 hr of delivery. The authors point out that while the exact dose and exposure interval are unknown, and no data conceming maternal/fetal cannabinoid ratios at peak blood concentrations are available, the data did demonstrate that cannabinoids are found

ET AL.

in fetal blood after maternal exposure. The present study was undertaken to provide time-course information on maternal plasma distribution, on the placental transfer and fetal distribution of THC, and on a major metabolite, 11 -nor-9-carboxy-THC ( 11-nor). METHODS Three-time mated 8- to lo-year-old female rhesus monkeys (Macncu mulutta, 8.5 to 10 kg) from the NCTR in-house breeding colony were utilized in this study. Their diet consisted of Purina monkey chow high protein jumbo biscuits provided twice a day and water ad libiturn. Fetal age was known within 248 hr. Maternal surgery was performed on a single day between 146 and 15 1 days of gestation (88-9 1% term). Food was withheld the evening prior to and the morning of surgery. On the morning of surgery anesthesia was induced in each animal with 10 mg/kg ketamine and maintained with gas anesthesia (halothane/N20/02). Following a laparotomy incision, a hysterotomy was performed and one intraplacental artery was cannulated in the direction of the primary disk as determined by the presence of fetal heart pulsations (Hill et al., 1980). A dose of 0.3 mg/kg THC was administered via a maternal radial vein. A dosing solution of 3.0 mg/ml (vehicle: saline/ethanol/Triton X100, 89/10/l by volume), yielding an injection volume of 0.1 ml/kg given as a bolus dose, was prepared from a stock solution of THC, 50 mg/ml in 100% ethanol sup plied by the National Institute of Drug Abuse (NIDA). Maternal blood samples (2 ml) were collected by venipuncture from a uterine vein at 0, 1, 3,6, 10, 1520, 30, 45, 60, 90, 120, and 180 min. Fetal blood samples (0.5 ml) were obtained from the intraplacental artery cannula at 1, 3,6, 15, 30,60, 120, and 180 min. Plasma was obtained from all blood samples immediately after collection and frozen at -70°C for later analysis. Fetuses were killed by exsanguination at 180 min, and fetal plasma, spleen, testis, lung, brain, liver, bile, kidney, adrenals, thymus, and placenta were collected and weighed. All fetal tissue samples were stored frozen at -7o’C until analysis. The plasma concentrations of THC and 1 l-nor were determined by radioimmunoassay (RIA). RIA kits were supplied by NIDA, and assayswere performed according to the instructions supplied in the kit. Standard and experimental plasma samples were extracted with methanol ( 100 pl plasma: 400 pl methanol). Fifty-microliter aliquots of the supematants were incubated with antiserum specific for THC and A8-‘2sI-THC for determination of THC plasma levels. Determination of 1 l-nor levels was performed by incubating 100-d aliquots of supematants with I l-nor antiserum and A*-‘*‘I-l l-nor. All incubation components were prepared according to kit instructions, and incubations were performed at 4°C overnight

FETAL

DISPOSITION

(at least 8 hr). A8-THC and A8-1 l-nor were used as the radioligands because they are more stable than THC or 1 l-nor and both bind to the antibody with similar affinities. Bound radioactivity was precipitated by immunobeads and counted on a Packard auto-gamma scintillation spectrometer. THC and 1 l-nor values for experimental plasma samples were interpolated from standard curves. Minimum sensitivities for THC and 1 l-nor in plasma were 2.5 and 2.0 rig/ml, respectively (90% binding). Precision (interassay coefficient of variation) for THC was 2.1% at 2.5 rig/ml and 5.8% at 50 rig/ml and for 1l-nor was 5.3% at 2 rig/ml and 5.4% at 100 @ml. Tissues were extracted using a modification of the methods of Schooler etal. (1976) and Alozie et al. (1980). Tissues were thawed and up to 500 mg was placed in a glass test tube containing 0.5 ml of methanol for each 100 mg of tissue. The tissue was homogenized with a Brinkman Polytron (type PT 10/35) using a setting of 7, and THC was extracted by vigorous shaking on a vortex mixer for three 30-set repetitions, three min apart. The samples were subsequently centrifuged at 5°C and 3000g for 10 min. The initial supematant was separated from the pellet and saved. The pellet was resuspended in 0.5 ml of methanol/ 100 mg of tissue, and the procedure was repeated. The second supematant was added to the first, resulting in a final extract concentration of 100 mg of tissue/ml of methanol. The supematants were stored at 5°C and assayed the following day. The recovery of THC by this procedure was determined by adding either A8?THC or A9-[3H]THC to untreated tissue samples before extraction and was found to be 9 l-95%. The concentrations of THC and 1 l-nor in tissues were determined by adapting the plasma assay kits for tissue determinations. THC- and 11-nor-specific antisera and A8-“‘1-THC and ‘25I-I l-nor from the kits were used. Standard curves were prepared with purified THC and I l-nor (supplied by NIDA) in methanol extracts oftissue (100 mg/ml) from untreated animals for each organ analyzed, except gall bladder and adrenals for which there was not enough tissue to prepare standards. THC and 1lnor levels in gall bladder and adrenals were determined by interpolation from spleen standard curves which had the median 50% binding ratio of all analyzed tissue. Minimum sensitivities for THC and 1 l-nor in tissue were 0.5 and 0.4 rig/g, respectively (90% binding). Precision (interassay coefficient of variation) for THC was 1.5% at 0.5 rip/g and 9.8% at 10 rig/g and for 1 l-nor was 2.0% at 0.4 rig/g and 7.6% at 20 ngig. Each sample was analyzed in duplicate. Intraassay coefficients of variation were less than 10% for THC and 11-nor. Samples with coefficients of variation between duplicates exceeding 10% were rejected and the tissue was reanalyzed. Student’s t test was used to test for significant differences in mean fetal tissue levels of THC (p < 0.05).

RESULTS The plasma time course of THC and 1 lnor after maternal iv dosing with 0.3 mg/kg

317

OF THC

.--. n-6 o-o .--.

hewnal A9-THC Fetal AO-THC Maternal 1 l-NOR Fetal 1 l-NOR

TME N MINUTES FROM DOSING

FIG. 1. THC and 1 l-nor plasma concentration (mean f SE) in three late-term rhesus monkeys given 0.3 mg/ kg THC iv.

THC is shown in Fig. 1 and Table 1. Maternal plasma THC levels were maximal at 3 min ( 14 19 + 156 rig/ml) and fetal plasma levels peaked at 15 min (83 + 12 rig/ml). The peak maternal plasma concentration of 11 -nor (44 + 18 rig/ml) occurred at about 1 hr, while no appreciable levels of 1 l-nor were detected in fetal plasma (~5 rig/ml in one fetus, no detectable levels in the other two). By 3 hr, maternal and fetal THC plasma levels were about equal (37 rig/ml). Fetal tissue concentrations of THC 3 hr after maternal dosing are shown in Fig. 2. Fetal thymus, adrenals, and bile had the greatest concentrations of THC. Only spleen tissue had a THC concentration lower than that found in plasma (21 f 8 rig/g vs 37 + 6 ng/ ml). The one placenta analyzed for THC and 1 l-nor concentrations contained 86 rig/g THC. 11-nor was not detected in the placenta or in any fetal tissue analyzed. The mean fetal brain THC concentration shown in Fig. 2 is the composite value of all brain areas as described in Fig. 3. Of the brain areas analyzed, THC levels tended to be the highest in the cerebellum, although there were no significant differences in THC levels among any brain areas.

318

BAILEY

ET

TABLE MATERNAL

0 1 3 6 10 15 20 30 45 60 90 120 180 1440 2880 4320 5760

No. 1 ***a -c 194 149 130 123 77

45 43 3 5 4 2

No.2

plasma

***

***

889 1263 485 440 419 265 217 131 71 57 44 31

854 1575 552 439 335 374 363 268 209 139 99

-

*** *** ***

2

3 40 66 62 54 47 41

1 73 77 85 88 55 37 35

Maternal

THC

No. 3

*** ***

Fetal plasma 1 3 6 15 30 60 120 180

I

AND FETAL PLASMA THC AND 11 -nor LEVELS (ngjml) AFTER IV ADMINISTRATION OF THC (0.3 mg/kg) IN THREE LATE-TERM RHESUS MONKEYS Maternal

Time (min)

AL.

2 6

Xk

SE

NDb 872+ 18 1419+156 41Ok 110 439k 1 301 f 80 256+ 71 2342 70 159k 57 140+ 70 98k 41 62+ 18 37k 6 2* 1 4f 1 ND ND

22 64 103 75 58 48 35

No. 2

No. 3

*** *** ***

*** *** ***

*** *** ***

2 19 49 51 55 45 76 65 55 29 2 4 2

12 23 33 31 28 25 15 12 10 9

4

***

*** 5

*** *** Fetal plasma

2+ 45+ 69+ 83-r72+ 56iz 44k 37-r-

1 15 4 12 10 2 4 2

11 -nor

No. 1

THC ***

plasma

*** *** *** *** *** 5 4 3

*** *** *** *** *** *** *** ***

11 23 30 40 40 34 27 27 *** 3

*** ***

Xk SE ND ND ND 6rt 3 14* 7 31* 11 35rt 8 38+ 9 37k 6 44+18 37k15 31+13 21+ 6 ND 4 + 0.6 ND ND

1 l-nor

*** *** *** *** *** *** *** ***

ND ND ND ND ND ND ND ND

’ ***, Below sensitivity of assay. b ND, not determined. ’ -, Coefficient of variation in assay > 10%.

DISCUSSION Peak measured fetal plasma THC levels were obtained 15 min after maternal iv dosing, indicating that THC rapidly crosses the placenta and enters the fetal circulation. THC, but not 11 -nor, was also detected in fetal tissues 3 hr after dosing. The finding that THC rapidly crosses the placenta is in close agreement with the observations of Idanpaan-Heikkila et al. (1969) who reported that after ip administration of 2 mg/kg [3H]THC to pregnant hamsters, ra-

dioactivity was present in the fetus within 15 min and peaked at 30 min. The earlier peak time observed in this study was probably due to the different route of administration. Substantial levels of THC were detected in fetal tissues 180 min after dosing. THC levels were highest in the thymus, adrenals, and bile. There are few literature reports available on THC levels in fetal tissues after maternal exposure to the compound and none available for primates. Whole body autoradiographic studies of pregnant mice (Freudenthal et al., 1972; Kennedy and Waddell, 1972; Ryrfeldt

FETAL

DISPOSITION

FIG. 2. Fetal tissue concentration ofTHC (mean f SE) 3 hr after maternal iv dosing with 0.3 mg/kg THC (n = 3 except for placenta where n = 1).

et al., 1973) have revealed that concentrations of radioactive cannabinoids in fetal tissues are generally low. The degree of sensitivity of these autoradiographic methods, however, did not allow for the determination of specific levels of THC in a particular fetal organ. Whole organ levels of radioactivity from THC and its metabolites have been determined in adult rats, mice, and rabbits after iv injection of radiolabeled THC or exposure to marijuana smoke. These studies reported the highest levels of THC in adrenals, bile, liver, lungs, and kidney. No other reports have indicated high levels of THC in the fetal thymus, although recent reports have suggested that a relationship exists between marijuana exposure and immune suppression (Klein et al., 1985). As maternal plasma levels of THC declined, maternal plasma levels of 1 l-nor, a major metabolite, increased. No appreciable levels of 11 -nor were detected in fetal plasma or tissues. The lack of 11 -nor in fetal plasma and tissue suggests that 1 l-nor, possibly because of its polarity, does not cross the placenta. This observation also suggests that the monkey fetus does not metabolize THC to 1 l-nor to an appreciable degree at this stage of development. Data regarding fetal distribution of THC in individual brain areas of primates are not available in the scientific literature. Although there were no significant differences among

OF THC

319

THC levels in the fetal brain areas we analyzed, THC concentrations were highest in cerebellum and brain stem and lowest in the frontal cortex. These data are similar to those reported by Dewey et al. (1973) in which adult pigeons were dosed with 10 mg/kg THC im and brain levels were analyzed 2.5 hr later. Layman and Milton (197 1) have also reported no significant selective distribution of THC in adult rat brain after ip administration of 10 mg/kg [14C]THC. However, 4 hr after dosing, they reported higher levels of radioactivity in cerebellum, medulla, and pons than in hippocampus or cerebral cortex. Other studies in adult animals including rats (Shannon and Fried, 1972), dogs (Martin et al., 1976), and squirrel monkeys (McIsaac et al., 197 1) have reported regional brain differences in THC distribution, mainly between gray and white matter. Possible reasons for inconsistencies in the data from the present study and earlier reports may be differences in species, dose, route of administration, and the time between dosing and examination. Also, fetal brain distribution may not be similar to that noted for adults. No llnor was found in any fetal brain areas examined in the current study. Marijuana smoke inhalation is the primary route of exposure for THC in the human population. Although this route was not evaluated in the present study of the pregnant rhesus monkey, other studies from our

FIG. 3. Fetal brain tissue concentration of THC (mean + SE) 3 hr after maternal iv dosing with 0.3 mg/kg THC (n = 3).

320

BAILEY

laboratory have compared THC plasma pharmacokinetics in the adolescent male monkey after iv THC and marijuana smoke exposure. The results indicate that a close similarity exists between THC plasma concentrations after a 0.3 mg/kg iv dose and 15 puffs of smoke (35 cm3 each) from a marijuana cigarette containing 2.85% THC delivered at a rate of three puffs per minute (Paule, Bailey, and Slikker, unpublished results). Although marijuana smoke inhalation studies in the pregnant monkey are yet to be completed, these comparisons in the adult suggest that iv values are predictive of those after relevant marijuana smoke exposures. Human time-course studies of the placental transfer of THC have not been reported. The present studies provide a predictive data base for human situations because of the numerous similarities between human pregnancy and rhesus monkey pregnancy. These similarities include functional hemochorial placentas, single births, and comparable maternal/fetal weight ratios (Reynolds et al., 1954; Chez and Hutchinson, 1969; Hendrickx and Sawyer, 1975; Novy et al., 1980). These data suggest that human exposure to marijuana during pregnancy would result in significant fetal exposure to THC. ACKNOWLEDGMENTS

ET AL. Bed A’-tetrahydrocannabinol in the rabbit. Biochem. 19, 1333-1339. ALOZIE, S. O., MARTIN, B. R., HARRIS, L. S., AND DEWEY, W. L. (1980). 3H-A9-tetrahydrocannabinol, 3H-cannabinol and ‘H-cannabidiol: Penetration and regional distribution in rat brain. Pharmacol. BioPharmacol.

them.

Behav.

12(2),

2 17-22

1.

BLACKARD, C., AND TENNES, K. ( 1984). Human placental transfer of cannabinoids. N. Engl. J. Med. 31 l( 12), 797.

CHEZ, R. A., AND HUTCHINSON, D. L. (1969). The use of experimental surgical techniques in the pregnant Macaca

mulatta.

Ann. N. Y. Acad. Sci. 162,249-253.

CHRISTENSEN, H. D., FREUDENTHAL, R. I., GIDLEY, J. T., ROSENFXLD, R., BOEGLI, G., TESTINO, L., BRINE, D. R., PITT, C. G., AND WALL, M. E. (197 1). Activity of A*- and A’-tetrahydrocannabinol and related compounds in the mouse. Science 172,165- 167. DEWEY, W. L., MCMILLAN, D. E., HARRIS, L. S., AND TURK, R. F. (1973). Distribution of radioactivity in brain of tolerant and nontolerant pigeons treated with 3H-A9-tetrahydrocannabinol. Biochem. Pharmacol. 22,399-405.

EVANS, M. A., AND HARBISON, R. D. (1977). Cocaine, marihuana, LSD: Pharmacological effects in the fetus and newborn. In Drug Abuse in Pregnancy, Neonatal Effects (J. L. Rementeria, Ed.), pp. 195-208. Mosby, St. Louis. FREUDENTHAL, R. I., MARTIN, J., AND WALL, M. E. ( 1972). Distribution of A9-tetrahydrocannabinol in the mouse. Brit. J. Pharmacol. 44,244-249. FRIED, P. A., BUCKINGHAM, M., AND KLUMIZ, P. V. (1983). Marihuana use during pregnancy and perinatal risk factors. Amer. J. Obstet. Gynecol. 146, 992994.

HARBISON, R. D., AND MANTILLA-PLATA, B. (1972). Prenatal toxicity, maternal distribution and placental transfer of tetrahydrocannabinol. J. Pharmacol. Exp. Ther. 180,446-453.

We thank Mr. Matthew Fogle and Mr. Michael Gillam for their technical assistance and Ms. Barbara Jacks for the typing of this manuscript. This work was supported in part by NIDA IAG 224-83-0005.

REFERENCES ABEL, E. L. (1980). Prenatal exposure to cannabis: A critical review of effects on growth, development and behavior. Behav. Neural Biol. 29, 131- 156. AGURELL, S., NILSSON, I. M., OHLSSON, A., AND SAND BERG, F. ( 1969). Elimination of tritium-labelled cannabinols in the rat with special reference to the development of tests for the identification of cannabis users. B&hem. Pharmacol. 18,l I95- 120 1. AGURELL, S., NILSSON, I. M., OHLSSON, A., AND SANBERG, F. (1970). On the metabolism of tritium-labe-

HARVEY, D. J. (1984). Chemistry, metabolism and pharmacokinetics of the cannabinoids. In Marihuana in Science and Medicine (G. G. Nahas, Ed.), pp. 37- 107. Raven Press, New York. HENDRICKX, A. G., AND SAWYER,R. H. (1975). Embryology of the rhesus monkey. In The Rhesus Monkey (G. H. Boume, Ed.), Vol. 2. pp. 141-169. Academic Press, New York. HILL, D. E., SLIKKER, W., JR., HELTON, E. D., LIPE, G. W., NEWPORT, G. D., SZISZAK, T. J., AND BAILEY, J. R. (1980). Transplacental pharmacokinetics and metabolism of diethylstilbestrol and 17@estradiol in the pregnant rhesus monkey. J. Clin. Endocrinol. Metab. SO,8 1 l-8 18. Ho, B. T., FFUTCHIE,G. E., KRALIK, P. M., ENGLERT, L. F., MCISAAC, W. M., AND IDANPAAN-HEIKKILA, J. (1970). Distribution of tritiated l-A9-tetrahydrocannabinol in rat tissues after inhalation. J. Pharm. Pharmacol.

22,538-539.

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DISPOSITION

J., FRITCHIE, Cl. E., ENGLERT, L. F., Ho, B. T., AND MCISAAC, W. M. ( 1969). Placental transfer of tritiated- 1-A9-tetrahydrocannabinol. N.

IDANPAAN-HEIKKILA,

Engl. J. Med.

281,330.

JONEIA, M. G. (1976). A study of teratological effects of intravenous, subcutaneous, and intragastric administration of A9-tetrahydrocannabinol in mice. Toxicol. Appl. Pharmacol. 36,15 I- 162. JONEJA, M. G. (1977). Effects of A’-tetrahydrocannabino1 on hamster fetuses. J. Toxicol. Environ. Health 2, 1031-1040. KENNEDY, J. S., AND WADDELL, W. M. (1972). Whole body autoradiography of the pregnant mouse after administration of “C-A9-tetrahydrocannabinol. Toxicol. Appl. Pharmacol.

22,252-258.

KLAUSNER, H. A., AND DINGELL, J. V. (197 1). The metabolism and excretion of A9-tetrahydrocannabinol in the rat. Lti Sci. 10,49-59. KLEIN, L. T. W., NEWTON, C. A., WIDEN, R., AND FRIEDMAN, H. (1985). The effect of A’-tetrahydrocannabinol and 11-hydroxy-A9-tetrahydrocatmabinol on T-lymphocyte and B-lymphocyte mitogen responses. J. Immunopharmacol.

I,45

I-466.

LAYMAN, J. M., AND MILTON, A. S. (197 1). Distribution of tritium labelled A’-tetrahydrocannabinol in the rat brain following intraperitoneal administration. &it. J. Pharmacol. 42,308-3 10. LEMBERGER, L., CRABTREE, R. E., AND ROWE, H. M. (1972). 1 I-Hydroxy-A’-tetrahydrocannabinol: Pharmacology, disposition, and metabolism of a major metabolite of marihuana in man. Science 177,62-64. MARTIN, B. R., DEWEY, N. L., HARRIS, L. S., AND BECKNER, J. C. (1976). 3H-A9-tetrahydrocannabinol tissue and subcellular distribution in the central nervous system and tissue distribution in peripheral organs of tolerant and nontolerant dogs. J. Pharmacol. Exp.

Ther. 196,128-144.

MCISAAC, W. M., FRITCHIE, G. E., IDANPAAN-HEIKIULA, J. E., Ho, B. T., AND ENGLERT, L. F. (1971). Distribution of marihuana in monkey brain and concomitant behavioral effects. Nature (London) 230, 593-594.

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321

MECHOULAM, R. (1970). Recent advances in cannabinoid chemistry opens the area to more sophisticated biological research. Science 168, 1159-I 166. NOVY, M. J., WALSH, S. W., AND COOK, M. J. (1980). Chronic implantation of catheters and electrodes in pregnant nonhuman primates. In Animal Models in Fetal Medicine (P. W. Nathanielsz, Ed.), Vol. 1, pp. 133- 168. Elsevier/North-Holland Biomedical Press, Amsterdam. PACE, H. B., DAVIS, W. M., AND BORGEN, L. A. ( 197 1). Teratogenesis and marijuana. Ann. N.Y. Acad. Sci. 191,123-132.

PERSAUD, T. V. N., AND ELLINGTON, A. C. (1968). The effects of cannabis sativa (Ganja) on developing rat embryos-Preliminary observations. West Indian Med.

J. 17,232-234.

REYNOLDS, S. R. M., PAULE, W. M., AND HUGCETT, A. ST. G. (1954). Physiological study of the monkey fetus in utero: A procedure for blood pressure recording, blood sampling, and injection of the fetus under normal conditions. Bull. Johns Hopkins Hosp. 95, 256268.

RYRFELDT, A., RAMSAY, C. H., NILSSON, I. M., WIDMAN, M., AND AGURELL, S. (1973). Whole body autoradiography of I-tetrahydrocannabinol and A’@)-tetrahydrocannabinol in mouse. Acta Pharmacol. Suet. 10, 13-28. SCHOOLER,J. C., Ho, B. T., AND ESTEREZ, V. S. (1976). Comparison of various solvent extractions for the chromatographic analysis of A9-tetrahydrocannabinol and its metabolites. In Marijuana: Chemistry, Biochemistry and Celhdar E&cts (G. G. Nahas, Ed.), pp. 63-69. Springer-Verlag, New York. SHANNON, M. E., AND FRIED, P. A. (1972). The macroand micro-distribution and polymorphic electroencephalographic effects of A9-tetrahydrocannabinol in the rat. Psychopharmacology27,141-156. VARDARIS, R. M., WEISZ, D. J., FAZEL, A., AND RAWITCH, A. B. (1976). Chronic administration of A9-tetrahydrocannabinol to pregnant rats: Studies of pup behavior and placental transfer. Pharmacol. Biochem. Behav.

4.249-254.