Development of an in vitro cytotoxicity model for aerosol exposure using 3D reconstructed human airway tissue; application for assessment of e-cigarette aerosol

AbstractDevelopment of physiologically relevant test methods to analyse potential irritant effects to the respiratory tract caused by e-cigarette aerosols is required. This paper reports the method development and optimisation of an acute in vitro MTT cytotoxicity assay using human 3D reconstructed airway tissues and an aerosol exposure system. The EpiAirway™ tissue is a highly differentiated in vitro human airway culture derived from primary human tracheal/bronchial epithelial cells grown at the air–liquid interface, which can be exposed to aerosols generated by the VITROCELL® smoking robot. Method development was supported by understanding the compatibility of these tissues within the VITROCELL® system, in terms of airflow (L/min), vacuum rate (mL/min) and exposure time. Dosimetry tools (QCM) were used to measure deposited mass, to confirm the provision of e-cigarette aerosol to the tissues. EpiAirway™ tissues were exposed to cigarette smoke and aerosol generated from two commercial e-cigarettes for up to 6h. Cigarette smoke reduced cell viability in a time dependent manner to 12% at 6h. E-cigarette aerosol showed no such decrease in cell viability and displayed similar results to that of the untreated air controls. Applicability of the EpiAirway™ model and exposure system was demonstrated, showing little cytotoxicity from e-cigarette aerosol and different aerosol formulations when compared directly with reference cigarette smoke, over the same exposure time

Corneal Epithelium Expresses a Variant of P2X7 Receptor in Health and Disease

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X7 form (defined as the canonical receptor) and its truncated forms. When Ca2+ mobilization is induced by BzATP, a P2X7 agonist, it is attenuated in the presence of extracellular Mg2+ or Zn2+, negligible in the absence of extracellular Ca2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death

Expression of receptor transcripts in epithelial cells.

<p><b>A.</b> RT-PCR was performed on HCLE mRNA showing presence of P2Y 1, 2, 4, 6, 11, and 12 receptor transcripts and P2X 4, 5, 6, and 7 receptor transcripts. GAPDH is included as internal control. <b>B.</b> PCR was performed on genomic DNA using exon specific primers. <b>C.</b> RT-PCR was performed on HCLE mRNA using primers that spanned exon 8 (to amplify variants f and j), showing an expected product size of 169 bp. GAPDH was amplified as an internal control. All products were sequenced and verified. Data is representative of a minimum of 3 independent experiments.</p

Expression of P2X₇ receptor changes with stratification.

<p><b>A.</b> Stratification was induced after day 4 (arrow) and cells were cultured for 4 additional days. Cells were lysed and equivalent amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-P2X₇. Representative blot is shown. Quantification of 75 kDa and 42 kDa protein are graphed as densitometric values relative to β-actin. Data is representative of a minimum of 4 independent experiments. <b>B.</b> Relative expression of P2X₇ mRNA was determined using the ΔΔCT method and the average of 3 independent cultures +/− SEM is presented. <b>C.</b> Apical cells in stratified culture of HCLE cells at day 7 prestimulated with BzATP show minimal uptake of ToPro-3 using the flow through aparatus. Cultures were prestimulated with BzATP for 5 minutes at which time ToPro-3 in the presence of BzATP was added. Cells were imaged for an additional 20 minutes in the presence of BzATP as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028541#pone-0028541-g004" target="_blank">Figure 4</a> (scale bar  = 50 µm). Arrow indicates representative areas of uptake. Control experiments were performed in the presence of HEPES buffer. <b>D.</b> Relative expression of Ki67 mRNA was determined using the ΔΔCT method and the average of 3 independent cultures +/− SEM is presented.</p

Diabetic corneal epithelium displays enhanced P2X₇ receptor expression compared to control non-diabetic corneal epithelium.

<p>Central corneal epithelium from 6 samples of control and diabetic were analyzed using real time PCR. Relative expression of the P2X₇ receptor (all variants), P2X_7j variant receptor, and Ki67 were determined using the ΔΔCT method. The average relative expression of 6 independent samples +/− SEM is presented. Significance was determined by Student's t-test: **p<0.002, *p<0.05 respectively.</p

HCLEs express a full length and truncated P2X₇ mRNA transcript and protein.

<p><b>A.</b> mRNA was purified from total RNA of HCLE and IMR90 cells cultured for 72 hours by twice passing over an oligo-dT column. Purity was confirmed by methylene blue staining of nytran filter following transfer (note lack of rRNA in mRNA lanes). <b>B</b>. Northern blot analysis of HCLE and IMR90 mRNA from 72 hour cultures. The probe used was generated by PCR amplification of a consensus region in all P2X₇ variants. The relative position of 18s rRNA is indicated and the smaller transcript expressed by epithelial cells is indicated (arrow). <b>C.</b> Expression of P2X₇ and P2X_7j mRNA transcript over a 72 hour incubation. Relative expression was determined using the ΔΔCT method. <b>D.</b> Expression of P2X₇ receptor by IMR90 cells. Cells were cultured for 72 hours, lysed and protein was resolved by SDS-PAGE (12%) and immunoblotted with anti-P2X₇. <b>E.</b> Expression of P2X₇ receptor by HCLE cells. Cells were cultured for 72 hours, lysed and protein was resolved by SDS-PAGE (12%) and immunoblotted with anti-P2X₇. Hetero- and homotrimers are identified on crosslinking gels. Epithelial cells were cultured for 72 hours, incubated in situ in the presence of formaldehyde, lysed and incubated at 65°C or 95°C to maintain or break crosslinks. Inset – equivalent experiment resolved by SDS-PAGE (8%) and immunoblotted with anti-P2X₇. Data is representative of a minimum of 3 experiments.</p

Cytotoxicity is not induced in epithelial cells following P2X₇ activation.

<p><b>A.</b> MTT assay for cell toxicity was performed on confluent HCLE cells. The response to control media, 100 µM BzATP or actinomycin D is presented as corrected absorbance (570nm–690nm). Data are representative of 3 independent experiments and are presented as +/− SEM. (Significance was determined by ANOVA followed by Tukey posthoc test; ** p<0.001. <b>B.</b> HCLE cells were stimulated with 100 µM BzATP, 2 µg/ml actinomycin D or control media lacking growth factors for 20 hr. Caspase activation was detected with NucView 488 Live Cell Caspase-3 Assay (scale bar  = 50 µm and applies to all 3 images). Images are representative of 5 independent experiments.</p