Xenobiotic metabolism in differentiated human bronchial epithelial cells

Pieter S. Hiemstra,Danyel G. J. Jennen,Binie Klein,Hans Gmuender,Jan J. W. A. Boei,Sylvia Vermeulen,Renate M. Verhoosel,Agustin Lahoz,Harry Vrieling

doi:10.1007/s00204-016-1868-7

Abstract

Differentiated human bronchial epithelial cells in air liquid interface cultures (ALI-PBEC) represent a promising alternative for inhalation studies with rodents as these 3D airway epithelial tissue cultures recapitulate the human airway in multiple aspects, including morphology, cell type composition, gene expression and xenobiotic metabolism. We performed a detailed longitudinal gene expression analysis during the differentiation of submerged primary human bronchial epithelial cells into ALI-PBEC to assess the reproducibility and inter-individual variability of changes in transcriptional activity during this process. We generated ALI-PBEC cultures from four donors and focussed our analysis on the expression levels of 362 genes involved in biotransformation, which are of primary importance for toxicological studies. Expression of various of these genes (e.g., GSTA1, ADH1C, ALDH1A1, CYP2B6, CYP2F1, CYP4B1, CYP4X1 and CYP4Z1) was elevated following the mucociliary differentiation of airway epithelial cells into a pseudo-stratified epithelial layer. Although a substantial number of genes were differentially expressed between donors, the differences in fold changes were generally small. Metabolic activity measurements applying a variety of different cytochrome p450 substrates indicated that epithelial cultures at the early stages of differentiation are incapable of biotransformation. In contrast, mature ALI-PBEC cultures were proficient in the metabolic conversion of a variety of substrates albeit with considerable variation between donors. In summary, our data indicate a distinct increase in biotransformation capacity during differentiation of PBECs at the air–liquid interface and that the generation of biotransformation competent ALI-PBEC cultures is a reproducible process with little variability between cultures derived from four different donors.

Highlights

Inhalation represents one of the major routes by which humans are exposed to gases, volatile compounds, aerosols and respirable particles
air–liquid interface (ALI)-PBEC were generated from primary bronchial epithelial cells derived from four donors
RNA was isolated from subconfluent cells grown on petri dishes under submerged conditions in KSFM medium, from cells grown to a confluent epithelial layer on tissue culture inserts in growth medium, just before or at various days (4, 7, 11, 14, 21 and 28) after initiation of air exposure during which the cell layer gradually differentiated into mucociliary epithelium

Summary

Introduction

Inhalation represents one of the major routes by which humans are exposed to gases, volatile compounds, aerosols and respirable particles. Cells of the respiratory tract have been shown to be metabolically competent and able to biotransform a large variety of inhaled substances (Anttila et al 2011; Hukkanen et al 2002; Zhang et al 2006). These biotransformation reactions enable detoxification of xenobiotics and facilitate elimination of hazardous compounds from the body (Nebert and Dalton 2006). In some cases biotransformation results in the formation of highly reactive intermediates that can bind to various biomolecules including DNA. A classic example is the activation of benzo(a)pyrene, a constituent of tobacco smoke and combustion products, into reactive species leading to the formation of mutagenic DNA adducts that have been implicated in the development of cancer (Denissenko et al 1996)

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