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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).
https://doi.org/10.1371/journal.pone.0185729.g001
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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.
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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.
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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.
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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.
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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 deficiency 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.
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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.
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S1 Fig. Landmark and measurements performed in the study.
(A) Landmarks (red dots) numbered in order of placement. (B) Measurements included face height (fh, green), intercanthal distance (id, red), dorsal mouth angle (dma, purple) and mouth roundness (mr, yellow dots outlining the mouth).
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S2 Fig. Whisker plots of the face measurements from embryos treated with “Lab Grade†e-cigAM.
Whisker plots are shown for intercanthal distance, face height, dorsal mouth angle and mouth roundness. The median is represented by the line through the box, the top whisker represents the maximum value and the bottom whisker represents the minimum value. Asterisks represent statistically significant differences.
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S3 Fig. Whisker plots of face measurements from embryos treated with flavored e-cigAM.
Whisker plots are shown for intercanthal distance, face height, dorsal mouth angle and mouth roundness for each e-cigAM A-F. The median is represented by the line through the box, the top whisker represents the maximum value and the bottom whisker represents the minimum value. Asterisks represent statistically significant differences.
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S4 Fig. Whisker plots of face measurements from embryos treated with e-cigAM E and F.
Whisker plots are shown for intercanthal distance, face height, dorsal mouth angle and mouth roundness in the comparison of e-cigAM E and F with and without nicotine. The median is represented by the line through the box, the top whisker represents the maximum value and the bottom whisker represents the minimum value. Asterisks represent statistically significant differences.
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