Fig 1.
Experimental setup for e-cigAM collection.
(A) Preparation of e-cigAM stock solution. ECIG vapor was aerated into 1 mL of 0.1X MBS by a 1.6 ohm atomizer attached to a syringe pump following a topography profile of: 3.5-second puffs (55 mL)with 30 second intervals between each puff for a series of 10. (B) Experimental set-up for X.laevis embryos. Embryos were arrayed in 6-well culture dishes in a 1:100 dilution of e-cigAM in 0.1X MBS. (C) E-cigAM exposure paradigm for qualitative and quantitative analysis. Embryos were exposed to e-cigAM from the 2-cell stage (1.5 hpf) to stage 43 (87 hpf).
Table 1.
The e-cigAMs tested on X. laevis embryos.
The nicotine concentration in mg/ml, ratio of propylene glycol to vegetable glycerin, and flavor additives are as reported on packaging.
Fig 2.
Effect of Lab Grade e-cigAMs on X. laevis orofacial development.
(A-K) Representative frontal views of (A) untreated control, (B-F) 1:10, or (G-K) 1:100 dilutions of lab grade e-cigAMs containing (B,G) 0 mg/ml, (C,H) 6 mg/ml, (D,I’) 12 mg/ml, (E,J) 18 mg/ml, or (F,K) 24 mg/ml concentration of nicotine. Mouth outlined in red dots. (L) Canonical variateanalysis of controls and 1:100 dilution of Lab Grade e-cigAMs. Black = untreated controls, pink = Lab Grade e-cigAM with 0 mg/ml nicotine, blue = Lab Grade e-cigAM with 18 mg/ml nicotine, green = Lab Grade e-cigAM with 24 mg/ml. Table includes Procrustes distances (PD) and p-values. Significant p-values in bold with asterisks. Wireframe graphs represent shape changes associated with position on graph. Canonical variate 1 = 66.0% variance, Canonical variate 2 = 21.5% variance. (M) Discriminant function analysis of controls compared to Lab Grade e-cigAM with (i) 0 mg/ml, (ii)18 mg/ml, or (iii) 24 mg/ml concentration of nicotine. Flat end of vector is average landmark position of controls, round end is average landmark position of Lab Grade e-cigAMs. Procrustes distances (PD) and p-values below graphs. (N) Measurements of intercanthal distance, face height, dorsal mouth angle, and mouth roundness of controls (black), and 1:100 dilutions of e-cigAMs containing 0 mg/ml (pink), 18 mg/ml (blue), or 24 mg/ml (green) concentration of nicotine. Controls were set to 100 and exposure groups normalized to it. Student’s t-test assuming unequal variance was performed on non-normalized data. Error bars represent standard error. Asterisks indicate significant difference when compared to controls. Alpha value for all statistical analyses = 0.02. cg = cement gland.
Fig 3.
Effect of flavored e-cigAMs on X. laevis orofacial development.
(A-G) Representative frontal views of (A) untreated controls, (B) embryos exposed to e-cigAM A, (C) embryos exposed to e-cigAM B, (D) embryos exposed to e-cigAM C, (E) embryos exposed to e-cigAM D, (F) embryos exposed to e-cigAM E, and (G) embryos exposed to e-cigAM F. Mouth outlined in red dots. (H) Canonical variate analysis of controls and e-cigAMs A-F. Black = untreated controls, red = e-cigAM A, purple = e-cigAM B, yellow = e-cigAM C, green = e-cigAM D, blue = e-cigAM E, pink = e-cigAM F. Wireframe graphs represent shape changes associated with position on graph. Canonical variate 1 = 68.8% variance, Canonical variate 2 = 15.8% variance. (I-N) Discriminant function analysis of controls compared to e-cigAMs A-F. Flat end of vector is average landmark position of controls, round end is average position of e-cigAMs. Procrustes distances (PD) and p-values below graphs. (O-T) Measurement of intercanthal distance, faces height, mouth angle, and mouth roundness of controls and e-cigAMs A-F. Controls were set to 100 and exposure groups normalized to it. Student’s t-test assuming unequal variance was performed on non-normalized data. Error bars represent standard error. Asterisks indicate significant difference when compared to controls. Alpha value for all statistical analyses = 0.02. cg = cement gland.
Table 2.
Procrustes distances and p-values derived from canonical variate analysis of controls compared to each e-cigAM exposure.
Fig 4.
Effect of flavored e-cigAMs with and without nicotine on X. laevis orofacial development.
(A,B) Representative frontal views of embryos exposed to (A) e-cigAM E- containing 0 mg/mL of nicotine or (B) e-cigAM E+ containing 18 mg/mL of nicotine. Mouth outlined in red dots. (C) Canonical variate analysis of controls (black), e-cig-AM E- (grey), and e-cigAM E+ (blue). Wireframe graphs represent shape changes associated with position on graph. Canonical variate 1 = 94.6% variance, Canonical variate 2 = 5.4% variance. (D) Discriminant Function analysis of e-cigAM E- compared to e-cigAM E+. Procrustes distances (PD) and p-values below graph. (E) Measurements of intercanthal distance, face height, mouth angle, and mouth roundness in e-cig-AM E- (grey), and e-cigAM E+ (blue). (F,G) Representative frontal views of embryos exposed to (F) e-cigAM F- containing 0 mg/mL nicotine or (G) e-cigAM F+ containing 6 mg/mL nicotine. (H) Canonical variate analysis of controls (black), e-cig-AM F- (grey), and e-cigAM F+ (pink). Wireframe graphs represent shape changes associated with position on graph. Canonical variate 1 = 79.8% variance, Canonical variate 2 = 20.2% variance. (I) Discriminant Function analysis of e-cigAM F- compared to e-cigAM F+. (J) Measurements of intercanthal distance, face height, mouth angle, and mouth roundness in e-cig-AM F- (grey), and e-cigAM F+ (pink). For bar graphs of size and shape measurements, controls were set to 100 and exposure groups normalized to it. Student’s t-test assuming unequal variance was performed on non-normalized data. Asterisks indicate significant difference when compared to controls. Alpha value = 0.02.
Fig 5.
A) Canonical variate analysis. Comparison of e-cigAM E with nicotine (e-cigAM E+) or without nicotine (e-cigAM E-) and e-cigAM F with nicotine (e-cigAM F+) or without nicotine (e-cigAM F-). Dark blue = e-cigAM E+, light blue = e-cigAM E-, dark pink = e-cigAM F+, light pink = e-cigAM F-. Wireframe graphs represent shape changes associated with position on graph. Table contains Procrustes distance (PD) and p-values. Significant p-values are bolded and denoted with an asterisk. Canonical variate1 = 74.2% variance, canonical variate 2 = 11.0% variance. B) Discriminant Function Analysis of (i) e-cigAM E- compared to e-cigAM F-, (ii) e-cigAM E+ compared to e-cigAM F+. Flat end of vector is average landmark position of either e-cigAM E- or e-cigAM E+, round end is average position of e-cigAM F- or F+. Red dots are landmarks with little to no obvious change in position. Alpha value for all statistical analyses = 0.02.
Fig 6.
e-cigAMs E and F affect jaw cartilage, muscle and cranial blood distribution.
(A-F) Cartilage labeling with Alcian Blue in representative embryos showing frontal views and lateral views in controls, e-cigAM E and F treated. Arrowheads are at right angles to the branchial cartilages. Arrows indicate the more obtuse angle of the infrarostral cartilages. (G-L) F-actin labeling of muscle (green) and Hoescht’s nuclear stain (blue) in representative embryos showing ventral views and frontal views in controls, e-cigAM E and F treated. Arrowheads indicate defects noted in the ih in I, and the lm in K and L. Arrows indicate defects in the gh in I J, K and L. M-R) Blood cell labeling with benzidine-peroxidase in blue of representative embryos showing frontal views and lateral views in controls, e-cigAM E and F treated. Arrows indicate deficicy in the mab in P and R. Abbreviations: ba’s = branchial cartilages, ch = cerathoyal, Mk = Meckel’s, ir = infrarostral, eth = ethmoid, qha = quadratohyoangularis, oh = orbitohyoideus, lm = levator mandibulae, ih = interhyoideus, gh = geniohyoideus, cbs = constrictors branchialium, mab = musculoabdominal vein, aa3 = third aortic arch, cg = cement gland.
Fig 7.
e-cigAM pertubs gene expression and cell health in murine neural crest cells.
A) Experimental design. B,C) qRT-PCR showing normalized expression levels of vegf, fgf2, sox9 and Col2a1 in cells treated with e-cigAM E (B) and e-cigAM F(C). D,E) Alamar Blue assay showing relative fluorescence after 24 and 72 hours e-cigAM E (B) and e-cigAM F (C) treatment. Asterisks indicate significance.