Effect of maternal immunopotentiation on apoptosis-associated molecules expression in teratogen-treated embryos.

PROBLEM: Potentiation of the maternal immune system was shown by us to affect the embryonic response to teratogenic insults. In order to understand better the mechanisms underlying that phenomenon, we explored the effect of maternal immunopotentiation by rat splenocytes on the early stages of the embryonic response to cyclophosphamide (CP). METHOD OF STUDY: Immunopotentiated CP-treated embryos were analysed for cell cycle changes by flow cytometry, while cell proliferation and apoptosis were assessed by 5'-bromo-2'-deoxyuridine (BrdU) incorporation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) respectively. The expression of the p65 subunit of NF-kappaB, IkappaBalpha, Bax, bcl-2 and p53 was assessed by flow cytometry. RESULTS: Exposure to CP resulted in significant growth retardation and in the appearance of cellular damage, a reduction in cell proliferation and the appearance of apoptotic cells, which were all found to be delayed in immunopotentiated embryos. In parallel, CP-treated embryos demonstrated a reduction in the percentage of p65- or IkappaBalpha-positive cells, while the percentage of bcl-2- or p53-positive cells increased initially and decreased later. Those changes were normalized by maternal immunopotentiation when tested at 24 hrs after exposure to the teratogen. CONCLUSION: Our data implicate maternal immunopotentiation to protect the embryo against teratogenic insults, possibly through its effect on the expression of p65, bcl-2 or p53.

Tumor necrosis factor-alpha-associated mechanisms affecting the embryonic response to cyclophosphamide.

PROBLEM: We have previously shown that TNF-alpha(-/-) embryos are more sensitive to the exposure to cyclophosphamide (CP) compared with TNF-alpha(+/+) embryos; however, the underlying mechanisms are not fully understood. Thus, in our present study, we tried to identify those molecules that might be responsible for the protective effect of the cytokine. METHOD OF STUDY: CP-treated TNF-alpha(-/-) and TNF-alpha(+/+) embryos were analyzed for changes in apoptosis by TUNEL and flow cytometry, while cell proliferation was analyzed by BrdU incorporation. The expression of Bax, bcl-2, p53, the p65 subunit of NF-kappaB and IkappaBalpha was assessed by Western blotting and immunohistochemistry. RESULTS: CP-treated TNF-alpha(-/-) embryos exhibited a more profound decrease in their weight, which was accompanied by an earlier appearance of cellular damage and apoptotic cells and an earlier decrease in cell proliferation in the embryonic brain compared with TNF-alpha(+/+) embryos. Also, an increased percentage of Bax-positive cells and a decreased percentage of bcl-2-positive cells were detected in TNF-alpha(-/-) embryos 48 hr after exposure, which were accompanied by a decreased percentage of p53-positive cells. CONCLUSION: Our data implicate TNF-alpha to be involved in the protection of the embryo against CP teratogenicity, possibly via alteration in Bax, bcl-2 or p53 expression.

Expression of apoptosis-associated molecules in the fetoplacental unit of cyclophosphamide-treated mice.

The mechanisms underlying the teratogen-induced apoptotic process leading to anomaly formation are not as yet understood. Therefore, we tried to evaluate possible changes in the expression of molecules regulating the apoptotic process induced in the embryo and placenta by exposure to cyclophosphamide (CP). Exposure to CP resulted in clear growth retardation that was accompanied by a time-dependent increase in cellular damage and an appearance of apoptotic cells in the embryonic brain and limbs as well as a decrease in cell proliferation. Western blot analysis demonstrated an increase in the level of Bax and a decrease in the expression of the p65 subunit of NF-kappaB and IkappaB alpha in the embryo and placenta. Immunohistochemical analysis localized cells expressing those molecules to the areas that exhibited CP-induced cellular damage, while in the placenta they were revealed mainly in the luminal and glandular epithelium. Our results suggest a possible involvement of Bax, p65 and IkappaB alpha in the response of the embryo and the placenta to teratogenic insults.

TNF-alpha protects embryos exposed to developmental toxicants.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) has been implicated in mediating post-implantation embryo loss or the embryonic maldevelopment induced by development toxicants or maternal metabolic imbalances. In order to clarify the role of TNF-alpha further, a comparative study was performed in TNF-alpha, knockout and TNF-alpha, positive mice, exposed to a reference teratogen, cyclophosphamide (CP). METHODS: Cyclophosphamide was injected on day 12 of pregnancy and 18-day fetuses were examined for external structural anomalies. Apoptosis and cell proliferation were measured by TdT-mediated biotin-dUTP nick-end labeling and 5'-bromo-2'-deoxyuridine incorporation, respectively, in the brain (an organ, sensitive to the teratogen) of embryos 24 hr after CP injection. NF-kappaB DNA-binding activity by electrophoretic mobility shift assay (EMSA) and the expression of Re1lA (an NF-kappaB subunit) and I(kappa)B(alpha) proteins by Western blot analysis were assessed in the brain of embryos tested 24 and 48 hr after CP treatment. RESULTS: Surprisingly, the proportion of fetuses with craniofacial, trunk and severe limb reduction anomalies were significantly higher in TNF-alpha -/- females, than in TNF-alpha,+/+ mice. Excessive apoptosis and suppression of cell proliferation was found in the brain, and they were more prominent in TNF-alpha -/- than TNF-alpha +/+ embryos, when examined 24 hr after CP injection. Finally, CP-induced suppression of NF-kappaB DNA-binding activity was found to be enhanced in the brain of TNF-alpha -/- embryos, and the restoration of NF-kappaB DNA-binding activity was compromised. CONCLUSION: This work demonstrates for the first time that TNF-alpha may act as a protector of embryos exposed to teratogenic stress. One possible mechanism may be restoration of NF-kappaB activity in embryonic cells surviving the teratogenic insult.

Potentiation of the maternal immune system may modify the apoptotic process in embryos exposed to developmental toxicants.

PROBLEM: We have previously shown that teratogen-induced embryonic maldevelopment may result from excessive apoptosis in affected organs, but the mechanisms underlying this process are not well understood. Here we investigate the ability of maternal immunopotentiation to affect the apoptotic process and its regulatory genes p53 and bcl-2 in embryos exposed to a teratogenic insult. METHOD OF STUDY: Potentiation of the immune system in pregnant females was performed with xenogeneic rat splenocytes or with granulocyte macrophage-colony stimulating factor (GM-CSF). The animals were exposed to cyclophosphamide (CP) and the reproductive performance in the various experimental groups was recorded. The level of apoptosis was assessed in the embryonic head and liver by TdT-mediated dUTP-biotin nick end labeling and fluorescence-activated cell sorter (FACS) analysis, while p53 and bcl-2 expression was evaluated by FACS and immunohistochemistry. RESULTS: In CP-treated females, a decrease in embryonic weight and an increase in the resorption rate and the percentage of embryos exhibiting head malformations were noted. These effects of CP were accompanied by the appearance of apoptotic cells in the head but not in the liver and an increased expression of p53 in embryonic organs, while bcl-2 expression was found to be decreased in the head and increased in the liver. Immunopotentiation with rat splenocytes or GM-CSF was shown to partially normalize the teratogenic effect of CP. It was also found to partially decrease the CP-induced apoptotic process and exhibited a tendency to normalize the expression of p53 and bcl-2 in the embryonic head and liver. CONCLUSION: Our results suggest a possible role for maternal immunopotentiation in protecting the embryo from teratogenic insults, possibly through regulation of the CP-induced apoptotic process and the expression of p53 and bcl-2.

NF-kappa B DNA-binding activity in embryos responding to a teratogen, cyclophosphamide.

BACKGROUND: The Rel/NF-kappaB transcription factors have been shown to regulate apoptosis in different cell types, acting as inducers or blockers in a stimuli- and cell type-dependent fashion. One of the Rel/NF-kappaB subunits, RelA, has been shown to be crucial for normal embryonic development, in which it functions in the embryonic liver as a protector against TNFalpha-induced physiological apoptosis. This study assesses whether NF-kappaB may be involved in the embryo's response to teratogens. Fot this, we evaluated how NF-KappaB DNA binding activity in embryonic organs demonstrating differential sensitivity to a reference teratogen, cyclophosphamide, correlates with dysmorphic events induced by the teratogen at the cellular level (excessive apoptosis) and at the organ level (structural anomalies). RESULTS: The embryonic brain and liver were used as target organs. We observed that the Cyclophosphamide-induced excessive apoptosis in the brain, followed by the formation of severe craniofacial structural anomalies, was accompanied by suppression of NF-kappaB DNA-binding activity as well as by a significant and lasting increase in the activity of caspases 3 and 8. However, in the liver, in which cyclophosphamide induced transient apoptosis was not followed by dysmorphogenesis, no suppression of NF-kappaB DNA-binding activity was registered and the level of active caspases 3 and 8 was significantly lower than in the brain. It has also been observed that both the brain and liver became much more sensitive to the CP-induced teratogenic insult if the embryos were exposed to a combined treatment with the teratogen and sodium salicylate that suppressed NF-kappaB DNA-binding activity in these organs. CONCLUSION: The results of this study demonstrate that suppression of NF-kappaB DNA-binding activity in embryos responding to the teratogenic insult may be associated with their decreased resistance to this insult. They also suggest that teratogens may suppress NF-kappaB DNA-binding activity in the embryonic tissues in an organ type- and dose-dependent fashion.