Effects of nitrofen and vitamins A, C and E on maturation of cultured human H441 pneumocytes.
OBJECTIVE
Nitrofen (2,4-dichloro-4 -nitrodiphenyl ether), a teratogen with oxidant properties, induces congenital diaphragmatic hernia (CDH) with lung hypoplasia and delayed lung development and maturation in rat embryos. Several phenotypic features of the alveolar epithelium including surfactant proteins A and B synthesis and its regulation by transcription factors are reproduced in cultured human H441 pneumocytes. The aim of the present study was to test whether vitamins A, E and C with anti-oxidant properties were able to recover the expression of such regulators in an in vitro setting.


METHODS
Cultured human H441 pneumocytes were treated with nitrofen with or without additional exposure to vitamins A, E and C. Thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3-beta (HNF-3beta) and hepatocyte nuclear factor 3-beta surfactant protein B (SP-B) mRNAs were measured by real-time polymerase chain reaction (RT-PCR). The cells were also immunohistochemically stained for assessment of proliferation (PCNA) and apoptosis (bis-benzimide) status and SP-B and TTF-1 protein expressions. Results were compared by ANOVA with a significant threshold of 5%.


RESULTS
Nitrofen severely decreased TTF-1, HNF-3beta and SP-B mRNA expression by H441 pneumocytes in culture. Addition of vitamin E normalized the levels of the three transcripts, while vitamin A normalized only those of TTF-1 and SP-B mRNA. Vitamin C was significantly beneficial only for SP-B transcript. Nitrofen decreased proliferation and TTF-1 and SP-B protein expressions with no apparent effect on apoptosis. Additional exposure to vitamins A, C or E rescued near normal values.


CONCLUSIONS
The changes induced by nitrofen in cultured H441 human pneumocytes are reverted in part by anti-oxidant vitamins by upregulating TTF-1, HNF-3beta and SP-B and stimulating proliferation and maturity in nitrofen-treated cells. These effects of anti-oxidant vitamins could be of some interest for developing new transplacental therapeutic strategies aimed at improving lung development and maturation in fetuses with CDH.
Gonzalez-Reyes
Salome
S
Department of Pediatric Surgery, Research Laboratory, Hospital Universitario La Paz, Madrid, Spain.
Martinez
Leopoldo
L
Martinez-Calonge
Wenceslao
W
Fernandez-Dumont
Virginia
V
Tovar
Juan A
JA
D001205:Ascorbic Acid / Q000494:pharmacology*; D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D051542:Hepatocyte Nuclear Factor 3-beta / Q000235:genetics; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010647:Phenyl Ethers / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D037701:Pulmonary Surfactant-Associated Protein B / Q000235:genetics; D014157:Transcription Factors; D014801:Vitamin A / Q000494:pharmacology*; D014810:Vitamin E / Q000494:pharmacology*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D010647:Phenyl Ethers; D037701:Pulmonary Surfactant-Associated Protein B; C504003:TTF1 protein, human; D014157:Transcription Factors; D014801:Vitamin A; D051542:Hepatocyte Nuclear Factor 3-beta; D014810:Vitamin E; C007350:nitrofen; D001205:Ascorbic Acid