Biological changes in C57BL/6 mice following 3 weeks of inhalation exposure to cigarette smoke or e-vapor aerosols

Figures & data

Table 1. Test materials and exposure concentrations.

Test materials	Materials or composition	Target exposure
		(µg/L TPM)	(µg/L Nicotine)
e-vapor*
Carrier	PG/VG/Nicotine (4% by weight)	1550	41
Test-1	Carrier + Flavors-1	1400	41
Test-2	Carrier + Flavors-2	1400	41
Cigarette
3R4F	Kentucky reference cigarettes	550	41
Control
Sham	Filtered Air	0	0

PG: propylene glycol; VG: vegetable glycerol.

* MarkTen^® (rechargeable e-cigarette with disposable pre-filled cartridges): battery length (67 mm), cartridge length (38 mm), diameter (9.2 mm), and coil resistance (3.5 Ohms) (Wagner et al., 2018).

Table 2. Exposure characterization.

Exposure parameter	Group
	Sham (Air) Control (0 µg TPM/L)	Carrier (1550 µg TPM/L)	Test-1 (1400 µg TPM/L)	Test-2 (1400 µg TPM/L)	3R4F (550 µg TPM/L)
Wet Total Particulate Matter (mean [µg/L]±SD) (%RSD)	0 ± 0 (NA)	1583 ± 44 (3%)	1410 ± 73 (5%)	1423 ± 54 (4%)	554 ± 16 (3%)
Carbon Monoxide (mean [ppm]±SD)	NM	NM	NM	NM	676 ± 23
Nicotine (mean [µg/L]±SD)	ND	43.1 ± 3.9	43.7 ± 2.5	43.7 ± 3.4	40.7 ± 2.8
Propylene Glycol (mean [µg/L]±SD)	ND	361 ± 21	311 ± 19	310 ± 26	ND
Glycerol (mean [µg/L]±SD)	ND	1058 ± 28	946 ± 26	960 ± 38	58.3 ± 5.0
Particle Size Distribution (MMAD [µM]±GSD)	ND	0.90 ± 1.6	1.0 ± 1.6	1.0 ± 1.7	0.70 ± 1.5
Acrolein (mean [µg/L]±SD)
Week 1	BFB	0.00190 ± 0.00023	0.00651 ± 0.00052	0.00308 ± 0.00068	3.28 ± 0.39
Week 3	BLOQ	BLOQ	BLOQ	BLOQ	3.45 ± 0.08
Acetaldehyde (mean [µg/L]±SD)
Week 1	BFB	0.00527 ± 0.00142	0.0358 ± 0.0058	0.0329 ± 0.0080	32.6 ± 0.5
Week 3	0.00490 ± NA	0.0286 ± 0.0024	0.0674 ± 0.0085	0.0641 ± 0.0183	34.9 ± 0.2
Formaldehyde (mean [µg/L]±SD)
Week 1	0.00921 ± 0.00699	0.0269 ± 0.0126	0.0336 ± 0.0057	0.0403 ± 0.0111	0.941 ± 0.103
Week 3	0.0216 ± 0.0148	0.192 ± 0.015	0.139 ± 0.028	0.145 ± 0.064	1.07 ± 0.13
Propionaldehyde (mean [µg/L]±SD)
Week 1	NA	0.00256 ± NA	0.00989 ± 0.00539	0.0123 ± NA	5.19 ± 0.28
Week 3	BFB	BFB	BFB	BFB	4.38 ± 0.17
Crotonaldehyde (mean [µg/L]±SD)
Week 1	ND	ND	ND	ND	1.77 ± 0.13
Week 3	BLOQ	BFB	BLOQ	BLOQ	0.787 ± 0.019

TPM: Wet total particulate matter; SD: Standard Deviation; RSD: Relative Standard Deviation; MMAD: Mass Median Aerodynamic Diameter; GSD: Geometric Standard Deviation; NA: Not applicable (in the case of SD, only two replicates were analyzed; no SD value generated); NM: Not Measured; ND: Not Detected; BFB: Below field blank concentration; BLOQ: Below lower limit of quantification.

TABLE 3. Biomarkers of exposure and respiratory function.

Parameter	Group
	Sham (Air) Control (0 µg TPM/L)	Carrier (1550 µg TPM/L)	Test-1 (1400 µg TPM/L)	Test-2 (1400 µg TPM/L)	3R4F (550 µg TPM/L)
Carboxyhemoglobin (mean [%]±SD)*
Day 1	0.7 ± 0.2	–^@	–	–	30.7 ± 4.2^a
Day 2	0.2 ± 0.3	–	–	–	38.5 ± 1.8
Day 3	0.6 ± 0.2	–	–	–	45.7 ± 0.6^a
Day 4	1.0 ± 0.3	–	–	–	45.2 ± 2.1^a
Day 5	1.0 ± 0.1	–	–	–	48.2 ± 0.9^a
Day 17	0.9 ± 0.5	1.3 ± 0.3^b	1.1 ± 0.1^b	1.5 ± 0.2^b^,^c	46.9 ± 1.9^a
Plasma Nicotine (mean [ng/mL]±SD)**
Day 17	<BLOQ(1.93)	1112 ± 1019^b	417 ± 57^b	506 ± 168^b	119 ± 11
Plasma Cotinine (mean [ng/mL]±SD)**
Day 17	<BLOQ(8.12)	3210 ± 1556^b	1940 ± 534^b	1730 ± 320^b	413 ± 36
Respiratory Physiology – Week 2 (mean ± SD; N = 6/group)
Respiratory Rate (breaths/min)	231.7 ± 17.7	174.6 ± 16.6^a^,^b	171.5 ± 9.9^a^,^b	193.2 ± 20.4^a^,^b^,^c	96.6 ± 19.1^a
Tidal Volume (mL/breath)	0.19 ± 0.02	0.22 ± 0.05	0.19 ± 0.03	0.21 ± 0.05	0.18 ± 0.05
Minute Volume (mL/min)	43.0 ± 4.3	39.1 ± 8.7^b	33.7 ± 6.0^b	39.7 ± 9.2^b	19.3 ± 6.6^a
Total Inhaled Mass – 1 hr (mg)^#	NA	3.67 ± 0.81^a^,^b	2.94 ± 0.53^a^,^b	3.51 ± 0.81^a^,^b	0.64 ± 0.22^a

* N = 3 all Groups; For Day 1–5, blood samples after an increasing exposure duration (0.5–4 hrs).

** N = 3 all groups except N = 1 for Sham (Air) Control Group.

@ Dash (–) indicates parameter not collected for this group at this time point.

^a p ≤ .05 compared to Sham (Air) Control.

^b p ≤ .05 compared to 3R4F.

^c p ≤ .05 compared to Test-1.

^# Total inhaled mass calculated using the actual TPM for this day over the 1-hour duration of exposure (for 4 hours duration (estimated) TIM would be 14.68, 11.76, 14.04, and 2.56 for the Carrier, Test-1, Test-2, and 3R4F groups, respectively).

BLOQ: Below Limit of Quantitation.

Figure 1. Bronchoalveolar lavage fluid (BALF) cytology. (A) Immunophenotyping count of (CD45+) leucocytes in BALF. (B) Immunophenotyping count of (CD3+) T-cells in BALF. The absolute numbers of CD45+ leucocytes or CD3+ T-cells in the BALF increased significantly in the 3R4F group compared to the Sham Control, but remained unchanged in the e-vapor groups. There were no significant differences between e-vapor groups. *Statistically different compared to the Sham Control (p<.05).

Figure 2. Volcano plots representing the differentially expressed genes in the lung. For each gene, the alteration in gene expression, calculated as the log2 fold-change, and plotted on the x-axis. The statistical significance (false discovery rate [fdr] < 0.05), proportional to the negative log10-adjusted p value, is plotted on the y-axis. Dots highlight genes that are statistically significantly up- and down-regulated (right and left quadrants, respectively) compared with sham.

Figure 3. Biological impact factor (BIF) and network perturbation amplitude (NPA). (A) BIF scores: the percentages give the relative biological impact, which is derived from the cumulated network perturbations caused by the treatment relative to the reference (the treatment showing the highest perturbation; here 3R4F as REF). Only the significant network perturbations are summarized further into this single number; hence, any component entering the score is significant by definition. For each treatment comparison, the δ value (−1 to 1) indicates how similar the underlying network perturbations are with respect to the reference. A value of 1 indicates that all the networks are perturbed by the same mechanisms. (B) Heatmap NPA scores: summary of subnetworks. A network is considered perturbed if, in addition to the significance of the NPA score with respect to the experimental variation, the two companion statistics (O and K), derived to inform on the specificity of the NPA score with respect to the biology described in the network, are significant. *O and K statistic p values below .05 and NPA significant with respect to the experimental variation.

Figure 4. Proteome set enrichment in the lung. Set enrichment was assessed by gene-set analysis (GSA). The fold-change compared with Sham Control is used as the protein-level statistic, the mean as the set statistic, and protein (Q1) and sample (Q2) permutation to assess statistical significance. Enrichment results are normalized per pathway, and the normalized GSA enrichment score is mapped to the color scale. Significance is indicated for the Q1 and Q2 tests in the figure, based on false discovery rate (fdr)-adjusted p values.

Table 4. Organ weights.

Parameter	Group*
	Sham (Air) Control (0 µg TPM/L)	Carrier (1550 µg TPM/L)	Test-1 (1400 µg TPM/L)	Test-2 (1400 µg TPM/L)	3R4F (550 µg TPM/L)
Organ weights — 3-Week sacrifice** (mean ± SD)
Brain (g)	0.4249 ± 0.0087	0.4238 ± 0.0182	0.4365 ± 0.0162^c	0.4209 ± 0.0099	0.4283 ± 0.0157
%Brain/Body Wt	2.072 ± 0.072	2.096 ± 0.179	2.055 ± 0.090	2.161 ± 0.205	2.131 ± 0.128
Adrenal gland (g)	0.009 ± 0.003	0.008 ± 0.002	0.009 ± 0.002	0.009 ± 0.003	0.008 ± 0.001
%Adrenal gland/Body Wt	0.043 ± 0.016	0.039 ± 0.014	0.044 ± 0.011	0.048 ± 0.014	0.038 ± 0.006
%Adrenal gland/Brain Wt	2.070 ± 0.803	1.858 ± 0.593	2.123 ± 0.456	2.218 ± 0.644	1.770 ± 0.296
Heart (g)	0.109 ± 0.010	0.108 ± 0.008	0.114 ± 0.007^c	0.104 ± 0.010	0.108 ± 0.013
%Heart/Body Wt	0.531 ± 0.054	0.532 ± 0.045	0.538 ± 0.046	0.535 ± 0.058	0.538 ± 0.073
%Heart/Brain Wt	25.644 ± 2.444	25.410 ± 1.717	26.174 ± 1.653	24.788 ± 2.082	25.274 ± 3.418
Liver (g)	0.987 ± 0.088	1.010 ± 0.072^b	1.007 ± 0.093^b	0.993 ± 0.138^b	0.874 ± 0.059^a
%Liver/Body Wt	4.803 ± 0.326	4.985 ± 0.386^b	4.732 ± 0.348^b	5.073 ± 0.660^b	4.336 ± 0.196^a
%Liver/Brain Wt	232.2 ± 20.4	238.6 ± 19.9^b	230.5 ± 18.0^b	235.7 ± 30.8^b	204.0 ± 13.7^a
Lungs (g)	0.155 ± 0.0.018	0.154 ± 0.010^b	0.160 ± 0.017^b	0.153 ± 0.012^b	0.194 ± 0.022^a
%Lungs/Body Wt	0.752 ± 0.074	0.760 ± 0.071^b	0.755 ± 0.079^b	0.787 ± 0.088^b	0.963 ± 0.106^a
%Lungs/Brain Wt	36.332 ± 3.817	36.245 ± 1.586^b	36.737 ± 3.801^b	36.451 ± 2.653^b	45.210 ± 4.413^a
Thymus Wt (g)	0.035 ± 0.009	0.028 ± 0.004^b	0.032 ± 0.008^b^,^c	0.022 ± 0.008^a^,^b	0.044 ± 0.007^a
%Thymus/Body Wt	0.172 ± 0.043	0.140 ± 0.024^b	0.151 ± 0.039^b^,^c	0.114 ± 0.037^a^,^b	0.220 ± 0.029^a
%Thymus/Brain Wt	8.314 ± 2.197	6.711 ± 1.084^b	7.356 ± 1.842^b^,^c	5.333 ± 1.832^a^,^b	10.351 ± 1.500^a

* N = 6 all groups.

** N = 12 all groups except N = 11 for Carrier Group.

^a p ≤ .05 compared to Sham (Air) Control.

^b p ≤ .05 compared to 3R4F.

^c p ≤ .05 compared to Test-2 (for organ weights Test-2 was used as the control for comparison to Test-1).

Table 5. Histopathology.

Parameter	Group
	Sham (Air) Control (0 µg TPM/L)	Carrier (1550 µg TPM/L)	Test-1 (1400 µg TPM/L)	Test-2 (1400 µg TPM/L)	3R4F (550 µg TPM/L)
Histopathology 3-Week Sacrifice
Nose/Turbinates – Number examined	12	11	12	12	12
Inflammation	0^a	0	0	0	7 (1.1)^b
Olfactory epithelium, Metaplasia	0	0	0	0	2 (1.0)
Respiratory epithelium, Erosion	0	0	0	0	11(1.0)
Respiratory epithelium, Metaplasia, Squamous epithelium	0	0	0	3 (1.0)	12 (1.3)
Respiratory epithelium, Regeneration	0	0	0	0	12 (1.0)
Larynx – Epiglottis – Number Examined	11	11	12	12	11
Inflammation	0	0	0	0	6 (1.0)
Respiratory epithelium, Metaplasia, Squamous epithelium	1 (1.0)	8 (1.0)	6 (1.0)	5 (1.2)	11 (1.9)
Hyperplasia of metaplastic epithelium	0	0	2 (1.0)	0	9 (1.4)
Larynx – Number Examined	12	11	12	12	11
Larynx – Lateral Wall
Hyperplasia	0	0	0	0	4 (1.5)
Inflammation	0	0	0	0	1 (1.0)
Metaplasia, Squamous cell	0	0	0	0	2 (2.0)
Ulcer	0	0	0	0	3 (1.7)
Larynx – Ventral Pouch
Hyperplasia	0	0	0	0	3 (1.7)
Metaplasia	0	0	0	0	1 (1.0)
Trachea – Number Examined	12	11	12	12	12
Erosion	0	0	0	1 (1.0)	0
Lung – Number Examined	12	11	12	12	12
Infiltrate, Cellular, Histiocytic	2 (1.0)	0	0	7 (1.0)	12 (1.7)
Infiltrate, Cellular Mixed, Multifocal	0	0	1 (1.0)	1 (1.0)	12 (1.1)

^a Number of tissues examined with lesion present.

^b Number in parentheses is the average severity (calculated by the sum of the severity scores divided by the incidence).

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