An assessment of vaping-induced inflammation and toxicity: A feasibility study using a 2-stage zebrafish and mouse platform

Abstract

It is currently understood that tobacco smoking is a major cause of pulmonary disease due to pulmonary/lung inflammation. However, due to a highly dynamic market place and an abundance of diverse products, less is known about the effects of e-cigarette (E-cig) use on the lung. In addition, varieties of E-cig liquids (e-liquids), which deliver nicotine and numerous flavor chemicals into the lungs, now number in the 1000s. Thus, a critical need exists for safety evaluations of these E-cig products. Herein, we employed a "2-stage in vivo screening platform" (zebrafish to mouse) to assess the safety profiles of e-liquids. Using the zebrafish, we collected embryo survival data after e-liquid exposure as well as neutrophil migration data, a key hallmark for a pro-inflammatory response. Our data indicate that certain e-liquids induce an inflammatory response in our zebrafish model and that e-liquid exposure alone results in pro-inflammatory lung responses in our C57BL/6J model, data collected from lung staining and ELISA analysis, respectively, in the mouse. Thus, our platform can be used as an initial assessment to ascertain the safety profiles of e-liquid using acute inflammatory responses (zebrafish, Stage 1) as our initial metric followed by chronic studies (C57BL/6J, Stage 2).

Keywords: C57BL/6J; Electronic cigarette (E-cig); Inflammation; Safety profile; Zebrafish; e-liquids.

Figure 1.. E-liquid exposure affects the survival of zebrafish embryos.

Survival rate of embryos after e-liquid exposure (Menthol, Virginia Tobacco (VT) or Mint) from 3 to 5 days-post-fertilization (dpf). CTL is embryo medium only. Sodium trimethylsilylpropanesulfonate (DSS) and trinitrobenzenesulfonic acid (TNBS) are known pro-inflammatory reagents. PG/VG is the vehicle control for the e-liquid. n = 8–10 embryos per group.

Figure 2.. Neutrophil migration assay in the Tg(lysC:dsRed) transgenic zebrafish.

(A) In the control, neutrophils are localized around the vascular niche of the caudal hematopoietic tissue (CHT) at resting state during normal development in transgenic Tg(lysC:dsRed) zebrafish. Activation of neutrophils, i.e., by the Mint e-liquid promotes migration from the vascular niche of the CHT, a well-known hallmark for a pro-inflammatory response. (B) Pattern analysis, i.e., the summation of all Nearest Neighbor Distances, is an indicator of dispersed neutrophils. Boxes in (A) indicate the posterior trunk/tail region of zebrafish embryos. (C) Quantification of the neutrophil migration assay data from panel (A). n = 10–12 zebrafish embryos per treatment group.

Figure 2.. Neutrophil migration assay in the Tg(lysC:dsRed) transgenic zebrafish.

(A) In the control, neutrophils are localized around the vascular niche of the caudal hematopoietic tissue (CHT) at resting state during normal development in transgenic Tg(lysC:dsRed) zebrafish. Activation of neutrophils, i.e., by the Mint e-liquid promotes migration from the vascular niche of the CHT, a well-known hallmark for a pro-inflammatory response. (B) Pattern analysis, i.e., the summation of all Nearest Neighbor Distances, is an indicator of dispersed neutrophils. Boxes in (A) indicate the posterior trunk/tail region of zebrafish embryos. (C) Quantification of the neutrophil migration assay data from panel (A). n = 10–12 zebrafish embryos per treatment group.

Figure 3.. E-liquid exposures result in pro-inflammatory responses in zebrafish embryos.

(A) Pro-inflammatory responses with increases in neutrophil migration in transgenic Tg(lyscC:dsRed) zebrafish embryos are observed after exposures (3 dpf to 5 dpf) to Virginia Tobacco (VT), Menthol and Mint e-liquids or the known pro-inflammatory chemicals DSS or TNBS. Fluorescent neutrophils were imaged and analyzed using ImageXpress Pico. (B) The cell distance indicates the total nearest neighbor distances from neutrophils in the posterior trunk/tail regions of the transgenic Tg(lysC:dsRed) zebrafish at 5 dpf. n = 10–12 zebrafish embryos per treatment group. # p<0.001; * p<0.0001.

Figure 3.. E-liquid exposures result in pro-inflammatory responses in zebrafish embryos.

(A) Pro-inflammatory responses with increases in neutrophil migration in transgenic Tg(lyscC:dsRed) zebrafish embryos are observed after exposures (3 dpf to 5 dpf) to Virginia Tobacco (VT), Menthol and Mint e-liquids or the known pro-inflammatory chemicals DSS or TNBS. Fluorescent neutrophils were imaged and analyzed using ImageXpress Pico. (B) The cell distance indicates the total nearest neighbor distances from neutrophils in the posterior trunk/tail regions of the transgenic Tg(lysC:dsRed) zebrafish at 5 dpf. n = 10–12 zebrafish embryos per treatment group. # p<0.001; * p<0.0001.

Figure 4.. Chronic vaping contributes to increased pulmonary pathology in mice infected with MHV-A59.

(A) Study design. (B) Inflammatory cytokine analysis of BAL fluid from mock-, PG/VG alone-, PG/VG + NIC alone-, “Mint” vape alone-, mock + MHV-, PG/VG + MHV-, PG/VG + NIC + MHV-, “Mint” vape + MHV-treated mice (compared with mock control). ANOVA was performed for multi-group comparisons (Tukey’s multiple comparison tests). Symbols and bars represent the mean ±SEM compared with the mock control (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001). N.S., not significant. (C) H&E staining of lung tissue sections isolated from mock-, PG/VG alone-, PG/VG + NIC alone-, “Mint” vape alone-, mock + MHV-, PG/VG + MHV-, PG/VG + NIC + MHV-, “Mint” vape + MHV-treated mice. Alveolar wall thickening and the infiltration of inflammatory cells into the interstitial spaces were particularly observable in the lungs from the MHV-alone and vape + MHV mice (indicated by arrows). 200× magnification. Images are representative of five images examined per lung. NIC = 33 mg/ml nicotine

Figure 4.. Chronic vaping contributes to increased pulmonary pathology in mice infected with MHV-A59.

(A) Study design. (B) Inflammatory cytokine analysis of BAL fluid from mock-, PG/VG alone-, PG/VG + NIC alone-, “Mint” vape alone-, mock + MHV-, PG/VG + MHV-, PG/VG + NIC + MHV-, “Mint” vape + MHV-treated mice (compared with mock control). ANOVA was performed for multi-group comparisons (Tukey’s multiple comparison tests). Symbols and bars represent the mean ±SEM compared with the mock control (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001). N.S., not significant. (C) H&E staining of lung tissue sections isolated from mock-, PG/VG alone-, PG/VG + NIC alone-, “Mint” vape alone-, mock + MHV-, PG/VG + MHV-, PG/VG + NIC + MHV-, “Mint” vape + MHV-treated mice. Alveolar wall thickening and the infiltration of inflammatory cells into the interstitial spaces were particularly observable in the lungs from the MHV-alone and vape + MHV mice (indicated by arrows). 200× magnification. Images are representative of five images examined per lung. NIC = 33 mg/ml nicotine

Figure 4.. Chronic vaping contributes to increased pulmonary pathology in mice infected with MHV-A59.

(A) Study design. (B) Inflammatory cytokine analysis of BAL fluid from mock-, PG/VG alone-, PG/VG + NIC alone-, “Mint” vape alone-, mock + MHV-, PG/VG + MHV-, PG/VG + NIC + MHV-, “Mint” vape + MHV-treated mice (compared with mock control). ANOVA was performed for multi-group comparisons (Tukey’s multiple comparison tests). Symbols and bars represent the mean ±SEM compared with the mock control (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001). N.S., not significant. (C) H&E staining of lung tissue sections isolated from mock-, PG/VG alone-, PG/VG + NIC alone-, “Mint” vape alone-, mock + MHV-, PG/VG + MHV-, PG/VG + NIC + MHV-, “Mint” vape + MHV-treated mice. Alveolar wall thickening and the infiltration of inflammatory cells into the interstitial spaces were particularly observable in the lungs from the MHV-alone and vape + MHV mice (indicated by arrows). 200× magnification. Images are representative of five images examined per lung. NIC = 33 mg/ml nicotine

Figure 5.

Model for the 2-Stage in vivo Screening Platform: Zebrafish to Mouse.