A Cannabinoid Analogue of D9-TetrahydrocannabinolDisrupts Neural Development in Chick
Delphine Psychoyos,1 Basalingappa Hungund,2,3,4 Thomas Cooper,2,3,4 and Richard H. Finnell1
1Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology, Texas A&M Health Science Center,Houston, Texas
2Department of Psychiatry, College of Physicians & Surgeons, Columbia University, New York, New York3New York State Psychiatric Institute, New York, New York
4Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York
Marijuana is the most commonly abused illicit drug by pregnant women. Its major psychoactive constituent, D9-THC(D9-tetrahydrocannabinol), crosses the placenta and accumulates in the f!tus, potentially harming its development. Inhumans, marijuana use in early pregnancy is associated with miscarriage, a fetal alcohol-like syndrome, as well aslearning disabilities, memory impairment, and ADHD in the offspring. Classical studies in the 1970 s have reacheddisparate conclusions as to the teratogenic effects of cannabinoids in animal models. Further, there is very little knownabout the immediate effects of D9-THC on early embryogenesis. We have used the chick embryo as a model in order tocharacterize the effects of a water-soluble D9-THC analogue, O-2545, on early development. Embryos were exposed tothe drug (0.035 to 0.35mg/ml) at gastrulation and assessed for morphological defects at stages equivalent to 914somites. We report that O-2545 impairs the formation of brain, heart, somite, and spinal cord primordia. Shorterincubation times following exposure to the drug show that O-2545 interferes with the initial steps of head process andneural plate formation. Our results indicate that the administration of the cannabinoid O-2545 during earlyembryogenesis results in embryotoxic effects and serves to illuminate the risks of marijuana exposure during thesecond week of pregnancy, a time point at which most women are unaware of their pregnancies. Birth Defects Res (Part B)83:477488, 2008. r 2008 Wiley-Liss, Inc.
Key words: cannabinoids; anencephaly; brain malformation; CNSdevelopment; neural plate; neural folds; neural tube defects; chick embryo;animal model
Marijuana is the most commonly abused illicit drug bypregnant women (NIDA, 2001; WHO, 1997). Its majorpsychoactive constituent, D9-THC (Gaoni and Mechou-lam, 1971), crosses the placenta and accumulates in thef!tus (Blackard and Tennes, 1984), potentially harmingits development. Marijuana use in early pregnancy isassociated with miscarriage (Day and Richardson, 1991),an increased prevalence of congenital malformations(Hecht et al., 1968; Carakushansky et al., 1969; Hingsonet al., 1982), as well as learning disability, memoryimpairment, and ADHD in the exposed offspring(Goldschmidt et al., 2004; Fried et al., 2005; Nolandet al., 2005). However, very little is known aboutmechanisms underlying the adverse effects of D9-THCin early pregnancy. Miscarriage itself could result fromseveral causes, including failure of blastocyst implanta-tion due to a nonreceptive endometrium, as demon-strated in rodent models (Paria et al., 1995), or secondaryto teratogenic effects during early organogenesis (weeks23 of human gestation). At such early stages, any severemalformations resulting from the marijuana exposure
would be lethal and result in spontaneous abortion, andthus the embryo would not be detectable for clinicalstudies. The adverse effects of marijuana use duringpregnancy are aggravated by the fact that the potency ofmarijuana preparations, in terms of contents of itspsychoactive constituent D9-THC, has increased nearly25-fold since 1970, when the content of D9-THC inmarijuana was 1.25%; it now reaches 15%30% in somepreparations (NIDA, 2001). Thus, the modern cannabissmoker may be exposed to doses of D9-THC many timesgreater than his or her counterpart in the 1960 s and 1970 s(WHO, 1997). This fact is important, because the effects ofD9-THC are dose related, and most of the research on
Published online in Wiley InterScience (www.interscience.wiley.com)DOI: 10.1002/bdrb.20166
Additional supporting information may be found in the online version ofthis article.
*Correspondence to: Delphine Psychoyos, Center for Environmentaland Genetic Medicine, Institute of Biosciences and Technology, TexasA&M Health Science Center, Houston, Texas 77030.E-mail: firstname.lastname@example.orgReceived 7 July 2008; Accepted 15 August 2008
Birth Defects Research (Part B) 83:477488 (2008)& 2008 Wiley-Liss, Inc.
cannabis was carried out in the 1970 s using doses thatreflected cannabis intake at the time (WHO, 1997).Classical studies in rodents show that the develop-
mental stage at which D9-THC is administered is acritical factor in determining the degree of D9-THCembryotoxicity. The period of greatest susceptibility tothe embryotoxic effects of D9-THC occurs during earlyorganogenesis (E6.08.0 in mouse). During this period,D9-THC administration results in a high incidence ofembryonic death and congenital malformations. Theincidence of fetal loss varies between 40% and 100%,depending on the route of administration, the species,
and the dosage used (Harbison and Mantilla-Plata, 1972;Mantilla-Plata et al., 1973; Uyeno, 1973; Fleischman et al.,1975; Wright et al., 1976). Surviving embryos presentwith malformations in the enteric and nervous systems(holoprosencephaly, anencephaly, exencephaly, cleft pa-late, degenerating spinal cord, and spina bifida), defec-tive skeletogenesis (absent or reduced sternebrae, extraor fused ribs, abnormal vertebral ossification) (Geber andSchramm, 1969; Mantilla-Plata et al., 1975; Jonega, 1976,1977; Rosenkrantz, 1978), and cognitive deficiencies inthe adult (Gianutsos and Abbatiello, 1972; Antonelli,2005). By contrast, exposure to D9-THC prior to
Fig. 1. Dose related effects of cannabinoid O-2545. (A) Structure of O-2545: The terminal carbon atom of the side chain of D9-THC issubstituted by an imidazole group in O-2545. This substitution results in a CB1 agonist which produces the same spectrum ofpharmacological effects in the mouse model as D9-THC. (BG) Representative embryos treated with vehicle alone (BD; PBS), or O-2545(+CB) at 0.035 mg/ml (E), 0.07 mg/ml (F), and 0.35 mg/ml (G). Embryos were treated at stages (HH) 4+ (B,E), 4 (C,F) and 4 (D,G).Control embryos reached the equivalent of stages 11 (B), 8+ (C), and 10 (D). Whole-mount in situ hybridization was performed withKrox20 (B,E), or Otx2 and Delta-1 (C,D,F,G). Red arrow in (E): failure of the neural tube to close in treated embryo (see text). All embryosare shown in dorsal view. Abbreviations: aip, anterior intestinal portal; fb, primordium for the forebrain; fg, margin of the foregut; hn,Hensens node; mb, primordium for the midbrain; hb, primordium for the hindbrain; nt, neural tube; psm, presomitic mesoderm; s,somites. Scale bar 500 mm in C,D,F,G; 400 mm in B,E.
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Birth Defects Research (Part B) 83:477488, 2008
organogenesis (peri-implantation stages), results in 100%embryo death before the embryo can reach organogen-esis (Paria et al., 1995; Persaud and Ellington, 1967,1968a,b). Administration of D9-THC after organogenesis(E1014) no longer results in a high incidence of death orcongenital malformations (Mantilla-Plata et al., 1975;Banerjee et al., 1975; Haley et al., 1975; Harbison et al.,1977; Fleischman et al., 1980).There is clearly a developmental period of suscept-
ibility to the embryotoxic effects of D9-THC. Thiswindow of susceptibility coincides with the period ofearly organogenesis. In the present studies, we havetargeted this developmental window using the chickembryo in culture model system (stages 31 to 12). Thissystem enables the continuous monitoring of drug-induced teratogenic effects over time. Thus, any mal-formations occurring during the initial stages of embryo-nic development can be easily detected without resultingin embryo death (Gebhardt, 1972; Kotwani, 1998).Furthermore, the separation of the embryo frommaternalinfluences permits the evaluation of a response attributedsolely to the effect of the drug applied to the embryo(Wilson, 1978; Jelinek, 1982). Finally, the chick embryorepresents a long-standing model of embryonic verte-brate development (Romanoff, 1960; Balinsky, 1975;Stern, 2002), with close similarities to the human embryoduring early organogenesis (Romanoff, 1960; Balinsky,1975; Rahilly and Muller, 1987; Sulik, 2008).D9-THC is highly lipophilic and thus requires solubi-
lisation with either DMSO or surfactant agents (Tween80t, Emulphort), all of which are potentially embry-otoxic. To overcome this problem, we have used a water-soluble analogue of D9-THC, termed O-2545 (Martinet al., 2006). In this cannabinoid, the terminal carbon ofthe side chain of D9-THC is substituted by an imidazolegroup (Fig. 1A). This substitution results in a cannabi-noid receptor 1 (CB1) agonist that produces the samespectrum of pharmacological effects in the mouse modelas D9-THC (Martin et al., 2006). Yet, O-2545 is soluble inPBS and thus readily applicable to the chick embryo inculture.
Chick Embryo Culture and Drug Administration
Chick embryos (Charles River, CT, USA) were ex-planted in culture (New, 1955; Psychoyos and Stern,1996a,b), and grouped according to developmental stage(Hamburger and Hamilton, 1951): group 1 (stage 31 to4; 39 controls, 63 treated), group 2 (stage 4 to 41; 39controls, 36 treated), group 3 (stage 5 to 6; 38 controls, 42treated) and group 4 (stage 7 to 8; 8 controls, 13 treated).These stages correspond to days 1319 (weeks 2 to 3) ofhuman pregnancy (Nishimura et al., 1974). Embryoswere treated with a single exposure of O-2545 in PBS(10 ml) at a given dose: Initially, O-2545 was solubilized inPBS