Bone loss in adult offspring induced by low-dose exposure to teratogens.

Maternal malnutrition during pregnancy was shown by numerous studies to result in the birth of offspring exhibiting altered bone characteristics, which are indicative of bone loss. We hypothesized that not only maternal malnutrition but also some developmental toxicants (teratogens) given at a dose inducing neither structural anomalies nor growth retardation can detrimentally affect skeletal health in adult offspring. To check this hypothesis, pregnant mice were exposed to a single injection of 5-aza-2-deoxycytidine (5-AZA) (a teratogen capable of inducing phocomelia of the hind limbs) at a sub-threshold teratogenic dose.

Teratogen-induced alterations in microRNA-34, microRNA-125b and microRNA-155 expression: correlation with embryonic p53 genotype and limb phenotype.

Background: In a large number of studies, members of the microRNA (miRNA)-34 family such as miRNA-34a, miRNA-34b, miRNA-34c, as well as miRNA-125b and miRNA-155, have been shown to be regulators of apoptosis. The ability of these miRNAs to perform this function is mainly attributed to their ability to interact with the p53 tumor suppressor, which is a powerful regulator of the teratologic susceptibility of embryos. We chose to explore whether miRNA-34a/b/c, miRNA-125b and miRNA-155 may play a role in teratogenesis by using p53+/- pregnant mice treated with cyclophosphamide (CP) as a model.

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.

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.

Teratogen-induced distortions in the classical NF-kappaB activation pathway: correlation with the ability of embryos to survive teratogenic stress.

Studies with diverse teratogens implicated the transcription factor NF-kappaB in mechanisms determining teratological susceptibility of embryos. Here, a teratogen such as cyclophosphamide (CP) was used to test whether teratogenic insult alters the classical NF-kappaB activation pathway, and how these alterations correlate with the ability of mouse embryos to resist the teratogen-induced process of maldevelopment. We observed that embryos tested 24 h after the exposure of females to 40 mg/kg CP exhibited a dramatic decrease in the level of NF-kappaB (p65 subunit)-DNA binding, IkappaB kinase beta (IKKbeta) activity, expression of p65 and IKKbeta proteins, as well as NF-kappaB inhibitory proteins (IkappaBs) such as IkappaBalpha, IkappaBbeta, and IkappaBepsilon, and died within the next 24 h.

p53 regulates cyclophosphamide teratogenesis by controlling caspases 3, 8, 9 activation and NF-kappaB DNA binding.

The tumor suppressor protein p53 regulates the sensitivity of embryos to such human teratogens as ionizing radiation, diabetes, and cytostatics. Yet, the molecular mechanisms whereby it fulfills this function remain undefined. We used p53 heterozygous (p53(+/-)) female mice mated with p53(+/-) males and then exposed to cyclophosphamide (CP) to test whether caspases 3, 8, and 9 and the transcription factor nuclear factor (NF)-kappaB may serve as p53 targets.

Diabetes-induced fetal growth retardation is associated with suppression of NF-kappaB activity in embryos.

Background: Mechanisms underlying diabetes-induced fetal growth retardation remain largely undefined. Two events such as the persistent activation of apoptosis or suppression of cell proliferation in embryos might directly result in fetal growth retardation. Evidence implicating the transcription factor NF-kappaB in the regulation of the physiological and teratogen-induced apoptosis as well as cell proliferation suggests that it may be a component of mechanisms underlying this pathology.

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.

Differential teratogenic response of TNFalpha+/+ and TNFalpha-/- mice to cyclophosphamide: the possible role of NF-kappaB.

Background: We observed previously that tumor necrosis factor alpha (TNFalpha)-knockout embryos are more sensitive to a cyclophosphamide (CP)-induced teratogenic insult than their TNFalpha-positive counterparts, implicating molecules acting in TNFalpha-activated antiapoptotic pathways in the mechanisms underlying this phenomenon. The main goal of this study was to assess whether the transcription factor nuclear factor kappaB (NF-kappaB) may be 1 of those molecules. Such a choice is based by evidence demonstrating TNFalpha as a powerful activator of NF-kappaB and a key role of the transcription factor in the most effective TNFalpha-activated antiapoptotic cascade.

Involvement of NF-kappaB in the response of embryonic cells to methotrexate.

The involvement of NF-kappaB in the regulation of the apoptotic process was demonstrated previously, however, its exact role has not been established yet. In order to unravel mechanisms underlying teratogen-induced cell death, we tried in our present study to assess the involvement of the p65 subunit of NF-kappaB in the response of mouse embryonic fibroblasts (MEFs) to the anti-cancer drug methotrexate (MTX), using p65 knockout MEFs (p65(-/-)). Indeed, this cell line was found to be more susceptible to the exposure to MTX, demonstrated by more profound changes in cell survival, cell cycle, proliferation and the percentage of apoptotic or necrotic cells, as compared to wild type (WT) MEFs.

Maternal immunopotentiation affects caspase activation and NF-kappaB DNA-binding activity in embryos responding to an embryopathic stress.

Problem: Increased embryonic resistance to teratogenic stresses as a result of maternal immunopotentiation is associated with a decrease in the intensity of teratogen-induced apoptosis in target embryonic structures. These findings suggest that this effect of maternal immunopotentiation might be realized through modification of the expression of molecules regulating the teratogen-induced apoptotic process. To examine this possibility, we evaluated caspases 3, 8 and 9 activation as well as nuclear factor (NF)-kappaB DNA-binding activity in the embryos of immunopotentiated mice exposed to cyclophosphamide (CP).

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.

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).

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.