Cannabinoids Exacerbate Alcohol Teratogenesis by a CB1-Hedgehog Interaction

Abstract

We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ⁹-THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.

Figure 1

Prenatal cannabinoid exposure causes eye and face malformations in fetal mice. (a) Representative photographs of GD 17 mice that had been exposed to CB treatments on GD 8, with eyes ranked in severity from normal (1) to anophthalmic (7). Minor alterations of pupil shape and size occur in ranks (2) and (3), while colobomas are scored as (4–6). (b) The incidence of defects in either the right or the left eye in fetal mice following GD 8 exposure to the CB vehicle (n = 63 fetuses/8 litters), HU-210 0.03 mg/kg (n = 89 fetuses/11 litters), HU-210 0.1 mg/kg (n = 35 fetuses/6 litters), CBD 1.7 mg/kg (n = 63 fetuses/8 litters), CBD 5.6 mg/kg (n = 60 fetuses/7 litters), CBD 17 mg/kg (n = 78 fetuses/10 litters), THC 0.56 mg/kg (n = 76 fetuses/9 litters), THC 1.7 mg/kg (n = 81 fetuses/11 litters), THC 5.6 mg/kg (n = 69 fetuses/10 litters), THC 17 mg/kg (n = 80 fetuses/10 litters). **p < 0.01, ***p < 0.001, Chi-Square vs. CB vehicle. (c–i) Craniofacial and palate malformations caused by prenatal CBs. (c) A typical face following CB vehicle treatment. (d) An exencephaly caused by HU-210 (0.1 mg/kg, 4 cases). (e) A philtrum deficiency caused by CBD (17 mg/kg, 1 case). (f) micrognathia caused by THC (17 mg/kg, 1 case, also observed after CBD 1.7 and 5.6 mg/kg). (g) Hematoxylin and eosin staining of coronal sections through the anterior palate of a typical CB vehicle-treated fetus. (h,i) Cleft anterior palates caused by THC (h 1.7 mg/kg, i 17 mg/kg), as indicated by the black arrows. Data are expressed as the percent of mice with eye defects (number of affected mice/total number of mice *100), observed in a single experiment. See also Supplementary Table 1 for data on eye defect severity, body weight and length, and the number of live offspring per litter. See Supplementary Fig. 2a,b for separate analysis of left and right eye defects.

Figure 2

Cannabinoids exacerbate alcohol-induced eye and face malformations. (a) The incidence of defects in either the right or the left eye in fetal mice following GD 8 exposure to the CB vehicle (n = 103 fetuses/13 litters), Alcohol 1.4 g/kg alone (n = 62 fetuses/10 litters), Alcohol 2.8 g/kg alone (n = 85 fetuses/10 litters), CP 55,940 0.25 mg/kg alone (n = 101 fetuses/12 litters), simultaneous CP 55,940 0.25 mg/kg + Alcohol 1.4 g/kg (n = 133 fetuses/16 litters), or simultaneous CP 55,940 0.25 mg/kg + Alcohol 2.8 g/kg (n = 50 fetuses/7 litters). (b) The incidence of defects in either the right or the left eye in fetal mice following GD 8 exposure to the CB vehicle (n = 103 fetuses/13 litters), HU-210 0.03 mg/kg alone (n = 89 fetuses/11 litters), simultaneous HU-210 0.03 mg/kg + Alcohol 1.4 g/kg (n = 122 fetuses/15 litters), THC 0.56 mg/kg alone (n = 76 fetuses/9 litters), or simultaneous THC 0.56 + Alcohol 1.4 g/kg (n = 87 fetuses/11 litters). Data are expressed as the percent of mice with eye defects (number of affected mice/total number of mice *100), observed in a single experiment. **p < 0.01, ***p < 0.001, vs CB vehicle; ^p < 0.05, ^^^p < 0.001 vs. respective doses of CP 55,940 alone using a Chi-square test. (c–k) Craniofacial, palate, and brain malformations following simultaneous alcohol and cannabinoids. (c,f,i) A typical fetal mouse face as well as hematoxylin and eosin staining of coronal sections through the anterior palate and the brain at the level of the septal area, following the CB vehicle treatment. (d,g,j) Anophthalmia and philtrum deficiency, as well as cleft palate (indicated by black arrows), holoprosencephaly, and septal deficiency caused by alcohol 1.4 g/kg + CP 55,940 0.25 mg/kg. (e,h,k) Near anophthalmia, philtrum, and nostril deficiency, as well as cleft palate and widespread brain structural changes caused by alcohol 1.4 g/kg + HU-210 0.03 mg/kg. See also Supplementary Table 2 for data on eye defect severity, body weight and length, and the number of live offspring per litter. See Supplementary Fig. 2c–f for separate analysis of left and right eye defects.

Figure 3

Cannabinoids exacerbate alcohol-induced eye and brain malformations in zebrafish. (a,b) The incidence of small eyes (a) and midbrain/hindbrain boundary defects (b) following exposure to fish water vehicle (n = 30 for eyes and MHB), 0.5% alcohol (n = 30 for eyes and MHB), 2.0% alcohol (n = 30 for eyes and MHB), CP 55,940 1.0 mg/L (n = 30 for eyes and MHB), CP 55,940 2.5 mg/L (n = 32 for eye, 34 for MHB), CP 55,940 3.8 mg/L (n = 41 for eye, 44 for MHB), CP 55,940 5.0 mg/L (n = 40 for eye, 43 for MHB), 0.5% alcohol + CP 55,940 1.0 mg/L (n = 32 for eye, 35 for MHB), 0.5% alcohol + CP 55,940 2.5 mg/L (n = 35 for eye, 45 for MHB), 0.5% alcohol + CP 55,940 3.8 mg/L (n = 44 for eye, 49 for MHB). MHB defects were measured following exposure from 5.25 to 24 hours post fertilization (hpf), and small eyes were measured following exposure from 5.25 to 48 hpf. **p < 0.01, ***p < 0.001, vs fish water vehicle, ^^p < 0.01, ^^^p < 0.001 vs. respective doses of CP 55,940 alone using a Chi-square or Fischer’s exact test, depending on sample size. Data are expressed as the percent of zebrafish with defects (number of affected zebrafish/total number of zebrafish *100), observed in a single experiment. Dotted lines reference the zero incidence of spontaneous eye and MHB defects. Dashed lines within the bars corresponding to the alcohol and CP 55,940 simultaneous treatment indicate the predicted additive effects. (c–h) Representative photographs of zebrafish eyes and midbrain/hindbrain boundaries (i–n) following vehicle (c,i), 0.5% alcohol (d,j), 2% alcohol (e,k), CP 55,940 2.5 mg/L (f,l), CP 55,940 5.0 mg/L (g and m), and 0.5% alcohol + CP 55,940 2.5 mg/L (h and n). The black line in (c–h) represents a 50 μm scale bar. The white arrows in (i,j,l) indicate the midbrain/hindbrain boundary.

Figure 4

Cannabinoids impair Sonic Hedgehog signaling. (a) Effects of different concentrations of CBD, CP 55,940 and HU-210 on Smoothened agonist, SAG, stimulation of Gli1-mediated firefly luciferase expression in ShhLight2 cells. All data are the mean (±SEM) ratio of Gli1-mediated expression of firefly luciferase to constitutive renilla luciferase from 6 samples conducted in triplicate in two independent experiments, normalized as a percent inhibition of the maximal SAG effect. (b) Effects of different concentrations of CP 55,940 on Gli1-mediated firefly luciferase induced by a maximally effective SAG concentration (300 nM) in ShhLight2 cells. All data are the mean (±SEM) ratio of Gli1-mediated expression of firefly luciferase to constitutive renilla luciferase from 5 samples performed in triplicate in a single experiment. (c) Effects of different concentrations of CBD (yellow), CP 55,940 (green) and HU-210 (black) on Gli1-mediated alkaline phosphatase expression induced by a maximally effective SAG concentration (100 nM) in C3H10T1/2 cells. All data are mean (±SEM) alkaline phosphatase expression from 3 replicate samples from a single experiment, normalized as a percent inhibition of the SAG effect. (d) Shh pathway gene expression as measured by qRT-PCR in mouse rostral neural tube, 24 hours following GD 8 exposure to the CB vehicle or CP 55,940 (0.25 mg/kg). Shh, Gli1, and Gli2 are normalized to the housekeeping gene 18 s and portrayed as log₂ fold change from vehicle. All samples were measured in triplicate and the data are portrayed from a single experiment. Individual embryos are portrayed as open or filled circles (n = 9 embryos/5 litters for vehicle for Shh, or 10 embryos/5 litters for Gli1 and Gli2 vehicle; n = 10 embryos/4 litters for CP 55,940), while means (±SEM) are portrayed as open or filled bars. *p < 0.05, **p < 0.01 using two-sided Student’s t-tests. Individual embryos are marked by different fill colors. (e) The incidence of small eyes and midbrain/hindbrain boundary defects in zebrafish embryos receiving exposure to Shh mRNA (N183) + fish water vehicle (n = 40 for eye, 45 for MHB), CP 55,940 2.5 mg/L alone (n = 32 for eye, 34 for MHB), Shh mRNA (N183) + CP 55,940 2.5 mg/L (n = 32 for eye, 41 for MHB), CP 55,940 5.0 mg/L alone (n = 54 for eye, 57 for MHB), Shh mRNA (N183) + CP 55,940 5.0 mg/L (n = 53 for eye, 46 for MHB), 0.5% alcohol alone (n = 41 for eye, 45 for MHB), Shh mRNA (N183) + 0.5% alcohol (n = 30 for eye and MHB), 0.5% alcohol + CP 55,940 2.5 mg/L (n = 35 for eye, 45 for MHB), Shh mRNA (N183) + 0.5% alcohol + CP 55,940 2.5 mg/L (n = 34 for eye, 45 for MHB). **p < 0.01, ***p < 0.001 vs Shh mRNA (N183) + vehicle, ^^p < 0.01, ^^^p < 0.001 vs corresponding CP 55,940 treatment in absence of Shh mRNA using a Chi-square test. Data are expressed as the percent of zebrafish with defects (number of affected zebrafish/total number of zebrafish *100) from a single experiment.

Figure 5

CB1 receptors mediate CB teratogenesis. (a) Effects of pretreatment with a CB1 receptor antagonist, SR 141716A, on CP 55,940-induced eye defects in mice. Mice received CB vehicle (n = 43/6 litters) SR 141716A 5.0 mg/kg + the CB vehicle (n = 62/8 litters), the CB vehicle + CP 55,940 0.5 mg/kg (n = 51/8 litters), SR 141716A 2.0 mg/kg + CP 55,940 0.5 mg/kg (n = 45/6 litters), or SR 141716A 5.0 mg/kg + CP 55,940 0.5 mg/kg (n = 88/10 litters). SR 141716A or CB vehicle was given 10 min before the second injection. **p < 0.01, ***p < 0.001 vs. CB vehicle, ^^p < 0.01 vs CP 0.5, using a Chi-square test. Data are the percent of mice with defects (number of affected mice/total number of mice *100). See Supplementary Table 4 for eye defect severity, body weight and length, and the number of live offspring/litter. See Supplementary Fig. 2a–f. for separate analysis of left and right eye defects. (b) Small eyes in zebrafish embryos receiving exposure to fish water vehicle (n = 39), SR 141716A 3.0 mg/L (n = 40), CP 55,940 5.0 mg/L alone (n = 54), SR 141716A 3.0 mg/L + CP 55,940 5.0 mg/L alone (n = 49), 0.5% alcohol + CP 55,940 2.5 mg/L (n = 42), SR 141716A 3.0 mg/L + 0.5% alcohol + CP 55,940 2.5 mg/L (n = 49). **p < 0.01, ***p < 0.001 vs. vehicle. ^^p < 0.01, ^^^p < 0.001 vs. corresponding treatment without SR 141716A using a Chi-square test. Data are the percent of zebrafish with defects (number of affected zebrafish/total number of zebrafish *100). (c,d) CB1-Smo co-localization in the mouse neural tube (GD 9.5) using a proximity ligation assay (Duolink) to visualize sites (fluorescent red) where anti-CB1 and anti-Smo antibodies bind within 40 nm of one another. (e,f). CB1-GPR 161 co-localization in the mouse neural tube (GD 9.5). Nuclei of neural tube cells are labeled with DAPI in blue. Sections (7 µm) were imaged on a confocal microscope using a 40× (c,e) and 63× (d,f) oil lens.

Figure 6

Schematic representation of hypothesized interactions between Smoothened and the CB1 receptor in the embryo. Left panel. During typical development, Sonic Hedgehog (Shh) alleviates the repression of Smoothened (Smo) by Patched (Ptch1) which allows Smo to translocate to the primary cilium. In the primary cilium, Smo, a G-protein coupled receptor, activates Shh pathway signaling, partly through its association with Gαi proteins. Gαi inhibits adenyl cyclase (AC) which, by inhibiting the conversion of adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP), inhibits the accumulation of protein kinase A (PKA) and prevents the proteolytic processing of Gli transcription factors into their repressor forms (Gli R). By maintaining the Gli activator (Gli A) state, the inhibition of PKA facilitates the gene transcription necessary for normal cell proliferation and development. Right panel. Alcohol (EtOH) and cannabinoids converge onto the Shh pathway to alter typical development. A mechanism by which alcohol affects development is through its inhibition of Shh, which reduces the number of Smo molecules at the primary cilia, and thereby reduces the activation of Shh signaling cascades. Cannabinoids, on the other hand, have two mechanisms of action. First, cannabinoids directly inhibit Smo and prevent the signaling of Smo through Gαi proteins, as described above. Additionally, cannabinoids stimulate CB1 receptors that, in the primary cilia, form heterodimers with Smo. CB1-Smo heterodimers associate with Gαs proteins, in addition to Gαi proteins. When Gαs signaling is activated by CB1 receptor agonism, PKA is stimulated which increases Gli R and decreases Gli A. Co-exposure to alcohol and cannabinoids therefore, inhibits Shh and Smo-Gαi signaling, while simultaneously stimulating CB1-Gαs signaling, and ultimately causing a greater reduction in Shh pathway activation than does exposure to either drug alone.