Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition

• Published in: Birth defects research. Part B. Developmental and reproductive toxicology 2010-06, Vol.89 (3), p.223-232
• Main Authors: Kling, David E., Cavicchio, Amanda J., Sollinger, Christina A., Schnitzer, Jay J., Kinane, T. Bernard, Newburg, David S.
• Format: Article
• Language: English
• Subjects: Aldehyde Dehydrogenase - antagonists & inhibitors; Animals; Antibodies; Apoptosis; Apoptosis - drug effects; Cell Line; Cell proliferation; Congenital defects; differentiation; diphenyl ether; DNA nucleotidylexotransferase; dose-response; Enzymes; Epithelial cells; Epithelium - drug effects; Epithelium - pathology; Explants; Fetuses; Gene expression; Gene Expression - drug effects; Genes, Reporter - genetics; Hernia; Histones; Humans; Isoenzymes - antagonists & inhibitors; Lipid Metabolism - drug effects; Lipids; Lung; Lung - drug effects; Lung - pathology; Mesenchyme; Mice; Phenyl Ethers - toxicity; Rats; Reporter gene; Response Elements - genetics; retinal; Retinal Dehydrogenase; retinaldehyde dehydrogenase; retinaldehyde dehydrogenase (RALDH); Retinoic acid; retinol; Staining and Labeling; teratogenesis; Tretinoin - pharmacology; vitamin A
• ISSN: 1542-9733; 1542-9741; 1542-9741
• DOI: 10.1002/bdrb.20247

BACKGROUND: Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling. METHODS: To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response‐element (RARE) was transfected into HEK‐293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace‐cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets. RESULTS: Nitrofen‐induced dose‐dependent declines in RARE‐reporter gene expression. However, similar reductions were observed in control‐reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen‐induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes. CONCLUSIONS: The observed declines in nitrofen‐associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition. Birth Defects Res (Part B) 89:223–232, 2010. © 2010 Wiley‐Liss, Inc.