Background/Aim: 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. Materials and 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-β (HNF-3β) and hepatocyte nuclear factor 3-β 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-3β 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-3β 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.

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Acute rejection after small bowel transplantation (SBTx) may facilitate bacterial translocation (BT) and subsequent changes in the liver, spleen, and lungs. This study investigated whether BT occurs after acute rejection and whether this is followed by changes in the structure of the intestine and the phagocytic organs interposed between the gut and the general circulation. Orthotopic SBTx was performed in allogeneic (ALLO) rat-strain combinations (BN-Wistar, n=5). For comparison we used syngeneic SBTx (SYN) (BN-BN, n=6) controls. Animals were sacrificed on postoperative day 7. Mesenteric lymph nodes and portal and caval blood were cultured for aerobes and anaerobes. Escherichia coli beta-galactosidase DNA was assessed by polymerase chain reaction in the blood samples. Intestine, liver, spleen, and lung protein and DNA contents were measured. Histologic changes were graded according to standard criteria of acute rejection. For comparisons we used chi(2) and nonparametric Mann-Whitney test with a threshold of significance of p<0.05. ALLO rats lost more weight after SBTx than SYN rats (-13.02+/-4.39% vs. -8.04+/-5.08% of preoperative weight), although the difference was not significant (ns). A variable degree of graft rejection was histologically demonstrated in all ALLO rats, and DNA/protein content in the graft was significantly higher in this group (0.245+/-0.85 vs. 0.134+/-0.21, p<0.05). Gram-negative enteric bacteria were found in 4/5 ALLO and 4/6 SYN rats (ns), and aerobic Gram-positive bacteria in 2/5 and 3/6 (ns), respectively. Anaerobic growth occurred in mesenteric lymph nodes in one ALLO rat and in the bloodstream in another one. E. coli DNA was isolated in none of the ALLO but in two SYN rats (ns). BT was frequent after SBTx in both syngeneic and allogeneic strain combinations. Contrary to our expectations, BT after SBTx was not higher in ALLO group rats. However, anaerobic germs were isolated only in this group.

Rats with experimental congenital diaphragmatic hernia (CDH) have heart hypoplasia and conotruncal and great vessel malformations that are likely related to disturbed neural crest developmental control. Neural crest cells communicate through intercellular gap junctions whose main protein is Connexin 43 (Cx43). The migration and participation of neural crest cells in heart development is likely influenced by this protein which might be also directly involved in myocardial development. Vitamin A is beneficial for heart hypoplasia in CDH rats. The aims of this study were to examine the status of Cx43 in the heart of embryonal rats exposed to nitrofen and to assess if vitamin A reverts these effects. Pregnant rats received either 100 mg nitrofen or olive oil on E9.5. Each group was divided into two subgroups according to the subsequent treatment with intragastric vitamin A (15,000 i.u.) or vehicle on E10.5 and E11.5. The pups were recovered on E13, E15, and E21 and the hearts were dissected out and pooled. Cx43 mRNA expression was determined by quantitative real-time PCR. Comparisons among groups were made with ANOVA and Bonferroni post hoc tests with a threshold of significance of P<0.05. In control rats Cx43 mRNA was minimally expressed on E13 and E15 and fully expressed on E21. Nitrofen significantly increased Cx43 mRNA on E15. Additional treatment with vitamin A tended to moderate this increase on E15. Cx43 was overexpressed in the hearts of nitrofen-exposed embryonal rats on day E15 of gestation. Vitamin A tended to normalize this expression. The mechanism of action of Cx43 deserves further investigation.

Rats with nitrofen-induced congenital diaphragmatic hernia (CDH) have heart hypoplasia and cardiovascular malformations. The mechanism of action of nitrofen involves changes in neural crest signaling. Pax3 function is required for cardiac neural crest cells to complete their migration to the developing heart. The aim of this study was to examine whether Pa x 3 expression is changed at two gestational endpoints in rat embryos or fetuses exposed to nitrofen. On day E9.5 of gestation, pregnant rats received either 100 mg of nitrofen (n=10) or vehicle alone (control, n=10). The fetuses were recovered on E15 or E21. Their hearts were dissected out and weighed. Pax3 mRNA expression was determined by real-time polymerase chain reaction. We used two-tailed Student's t-tests to compare groups, with a threshold of significance of p<0.05. Compared with controls, nitrofen-exposed fetuses had heart hypoplasia in terms of heart/body weight ratio (0.62+/-0.10% vs. 0.77+/-0.17%, p<0.05). Pax3 mRNA expression in the heart was significantly decreased on E15 in nitrofen-treated embryos (32.94+/-17.11 U vs. 55.09+/-11.56 U, p<0.05), and it was still decreased, although not significantly, in the hearts of nitrofen-exposed fetuses recovered on E21 (15.67+/-5.56 U vs. 20.51+/-5.92 U, not significant). In conclusion, Pax3 is underexpressed in the hearts of nitrofen-exposed embryonal rats before the end of gestation. The mechanism of action of Pax3 should be further investigated because it could be one of the targets for future prenatal transplacental intervention.