The involvement of NF-kappa B in the regulation of teratogen-induced apoptosis has not been established yet. Therefore, we tried to assess the involvement of the p65 subunit of NF-kappa B in the embryonic response to the anti-cancer drug Doxorubicin (DOX). Thus, exposure of p65 knockout (p65(-/-)) or wild type (WT) mouse embryonic fibroblasts (MEFs) to DOX resulted in a decrease in cell survival, culture density and cell proliferation, which was found to be more prominent in p65(-/-) MEFs. Those phenomena were accompanied by a DOX-induced increase in the proportion of apoptotic cells, which was demonstrated only in p65(-/-) cells and a G(2)/M arrest, which was found to be more prominent in WT cells. Furthermore, DOX-treated WT and p65(-/-) MEFs differed in their expression of various apoptosis-associated molecules, when the former demonstrated a decrease in the percentage of p65-positive and a more prominent decrease in the percentage of p53-positive cells, while a decreased percentage of ham-positive and a more prominent decrease in the percentage of bcl-2-positive cells was detected among the latter. The fact that the response of the cells to the teratogen was clearly p65-dependent implicates this molecule to be involved in the response of the embryonic cells to DOX. (C) 2012 Elsevier Ltd. All rights reserved.

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. Micro-computed tomography scanning revealed that femora of 5-month-old male offspring exposed in uterus to 5-AZA had trabecular microarchitecture indicative of bone loss. Furthermore, exposure to 5-AZA increased the susceptibility of offspring to postnatal chronic mild stress, which has been shown to induce bone loss in mice. While exploring possible mechanisms underlying this phenomenon, we observed that the expression of some microRNAs, which have been demonstrated as regulators of key osteoblastogenic genes, was altered in hind limb buds of embryos exposed to 5-AZA. Furthermore, the expression of receptor activator of nuclear factor kappa B ligand (RANKL) in femoral stromal/osteoblastic cells of 5-month-old offspring of 5-AZA-treated females was found to be increased. Collectively, this study implies for the first time that single low-dose exposure to a teratogen can induce bone loss in adult offspring, possibly via alteration of embryonic microRNAs and RANKL expression.

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. We evaluated how CP-induced alterations in the expression of these miRNAs in the embryonic limbs correlate with embryonic p53 genotype and CP-induced limb phenotypes. Results The limbs of p53 positive embryos were more sensitive to CP-induced teratogenic insult than the limbs of p53 negative embryos. The hindlimbs were more severely affected than the forelimbs. Robust miRNA-34a expression was observed in the fore- and hindlimbs of p53 +/+ embryos exposed to 12.5 mg/kg CP. The dose of 20 mg/kg CP induced almost a two-fold increase in the level of miRNA-34a expression as compared to that exhibited by p53 +/+ embryos exposed to a lower dose. Increased miRNA-34b and miRNA-34c expression was also observed. Of note, this dose activated miRNA-34a and miRNA-34c in the forelimbs of p53 -/- embryos. When embryos were exposed to 40 mg/kg CP, the expression pattern of the miRNA-34a/b/c was identical to that registered in the limbs of embryos exposed to 20 mg/kg CP. However, this dose suppressed miRNA-125b and miRNA-155 expression in the fore- and hindlimbs of p53 +/+ embryos. Conclusion This study demonstrates that teratogen-induced limb dysmorphogenesis may be associated with alterations in miRNA-34, miRNA-125b and miRNA-155 expression. It also suggests for the first time that p53-independent mechanisms exist contributing to teratogen-induced activation of miRNA-34a and miRNA-34c. At the same time, teratogen-induced suppression of miRNA-125b and miRNA-155 expression may be p53 dependent. The analysis of correlations between the expression pattern of the tested miRNAs and CP induced limb phenotypes implies that miRNAs regulating apoptosis may differ from each other with respect to their functional role in teratogenesis: some miRNAs act to protect embryos, whereas other miRNAs boost a teratogen-induced process of maldevelopment to induce embryonic death.

Whether the embryo develops normally or not depends not only on the mechanisms regulating embryonic development, but also on the mechanisms acting to resist and repair injures in the embryo due to harmful maternal stimuli or exposure to developmental toxicants. The key role of p53 in the regulation of the embryo's response to embryopathic stress inducing DNA damage is beyond doubt. Yet, the question why p53 in some cases acts as a suppressor of teratogenesis, whereas in other cases it induces teratogenesis, remains unanswered. In this minireview we analyze studies in which organogenesis-stage embryos were exposed to various developmental toxicants and suggest a model unifying the teratogenesis-suppressing and teratogenesis-promoting role of p53. This model predicts that p53 protects embryos from developmental toxicant inducing oxidative stress and promotes the process of maldevelopment induced by developmental toxicants activating apoptotic machinery. Certainly, many questions must be answered before concluding the extent to which this model is correct. Yet, it does allow us to explain some discrepancies obtained in studies performed to date. Also, the model might be useful in choosing molecular targets for further studies addressing p53-controlled and p53-independent mechanisms, which determine the embryo's resistance to embryopathic stress.

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.

Problem We have previously shown that TNF-α −/− embryos are more sensitive to the exposure to cyclophosphamide (CP) compared with TNF-α +/+ 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-α −/− and TNF-α +/+ 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-κB and IκBα was assessed by Western blotting and immunohistochemistry. Results CP-treated TNF-α −/− 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-α +/+ embryos. Also, an increased percentage of Bax-positive cells and a decreased percentage of bcl-2-positive cells were detected in TNF-α −/− embryos 48 hr after exposure, which were accompanied by a decreased percentage of p53-positive cells. Conclusion Our data implicate TNF-α to be involved in the protection of the embryo against CP teratogenicity, possibly via alteration in Bax, bcl-2 or p53 expression.

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.

Bax was shown previously to regulate apoptotic cell death in various experimental systems, however, its involvement in teratogen-induced apoptosis is not clear yet. Therefore, we explored the involvement of Bax in the response of mouse embryonic fibroblasts (MEFs) to the anti cancer drug methotrexate (MTX), using Bax wild type (WT) and knockout (Bax−/−) MEFs. Our results demonstrated a significant teratogen-induced dose- and time-dependant decrease in the survival and culture density of both cell lines, which were found to be somewhat more prominent in WT cells. Exposure to MTX resulted also in decreased cell proliferation of WT but not Bax−/− cells and accordingly, we observed an accumulation of cells in the S phase and an increased percentage of cells in the Sub-G1 phase of the cell cycle and the appearance of condensed nuclei, which were found to be somewhat more prominent in WT MEFs. In parallel, WT MEFs demonstrated a MTX-induced increase in the percentage of Bax-positive cells and a significant decrease in the percentage of bcl-2-, p65- or IκBα-positive cells, which were not detected in Bax−/− MEFs. Altogether, the differential sensitivity of WT or Bax−/− MEFs to MTX suggests a possible involvement of this molecule in the response of embryonic cells to teratogens.

Studies with diverse teratogens implicated the transcription factor NF-kappa B 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-kappa B 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-kappa B (p65 subunit)-DNA binding, I kappa B kinase beta (IKK beta) activity, expression of p65 and IKK beta proteins, as well as NF-kappa B inhibitory proteins (I kappa Bs) such as I kappa B alpha, I kappa B beta, and I kappa B epsilon, and died within the next 24 h. Embryos of females exposed to 15 mg/kg CP exhibited only a decrease in NF-kappa B-DNA binding and IKK beta activity at 24 h. However, at 48 h, a more prominent decrease in NF-kappa B activity was observed, accompanied by a decreased expression of p65 and IKK beta proteins. These embryos died within the next 24 h. After treatment with 10 mg/kg CP, embryos survived until the end of the antenatal period of development, demonstrating a transient decrease in NF-kappa B-DNA binding activity and no alterations in NF-kappa B signaling. These results suggest that the classical NF-kappa B activation pathway may be among targets that teratogens engage to initiate abnormal development. Besides, the observation that embryos destined to be dead exhibited a dramatically decreased rate of cell proliferation suggests a pathway, whereby teratogen-induced alterations in NF-kappa B signaling may culminate in such a final effect as embryonic death. (C) 2008 Published by Elsevier Inc.

Hyperglycemia-induced increase in the production of reactive oxygen species (ROS) is proposed to be an initial step in the pathogenesis of diabetes-induced spontaneous abortions and structural inborn anomalies. However, the subsequent steps in this process are incompletely understood. One of the key molecules involved is tumor necrosis factor-alpha (TNFalpha): its expression is regulated by ROS and it regulates ROS production in turn. This cytokine has been the focus of many studies addressing the mechanisms of different forms of diabetes-induced embryopathies, such as early pregnancy loss, inborn anomalies, fetal growth retardation as well as some pathologies appearing during adult life. In this review, we analyze the results of these studies and discuss how TNFalpha may regulate the response of pre- and post-implantation stage embryos to diabetes-induced detrimental stimuli. The data presented in this review suggest that TNFalpha may play a dual role in the pathogenesis of diabetes-induced embryopathies. It may act both as a mediator of diabetes-induced embryotoxic stimuli leading to the death of peri-implantation stage embryos and, possibly, as a suppressor of diabetes-induced apoptosis in post-implantation stage embryos. It also appears that TNFalpha fulfills these functions via interaction with leukemia inhibitory factor (LIF) and the transcription factor NF-kappaB. These molecules are presently considered as attractive targets for the treatment of diabetes-induced complications. Therefore, further studies addressing their role in the mechanisms underlying diabetes-induced embryopathies are needed to evaluate the safety of such therapies for diabetic women of childbearing age.

It was shown in the 1980s and early 1990s that fetomaternal immune interactions are important determinants influencing the embryo's development. Extreme complexity of the immune response, its feedback link with the neuroendocrine system and the striking diversity of processes that are dependent on or controlled by immune substrates during the embryo's development suggest that the genetically determined immune response may be one of the intrinsic factors determining the tolerance to teratogens as well. This study examined the evidence for such a hypothesis and some problems of teratological screening arising in the light of this evidence.

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. Mice were exposed to CP on day 12 of pregnancy and killed on days 15 and 18 of pregnancy to evaluate CP-induced teratogenic effect. The brain and limbs of embryos harvested 24 h after CP treatment were used to evaluate NF-kappaB (p65) DNA-binding activity by an ELISA-based method, the activity of the caspases by appropriate colorimetric kits, apoptosis, and cell proliferation by TUNEL, and 5'-bromo-2'-deoxyuridine incorporation respectively. We observed that the activation of caspases 3, 8, and 9 and the suppression of NF-kappaB DNA binding following CP-induced teratogenic insult took place only in teratologically sensitive organs of p53(+/+) but not p53(-/-) embryos. CP-induced apoptosis and suppression of cell proliferation were also more intensive in the former, and they exhibited a higher incidence of structural anomalies, such as open eyes, digit, limb, and tail anomalies. The analysis of the correlations between the p53 embryonic genotype, the activity of the tested molecules, and the CP-induced dysmorphic events at the cellular and organ level suggests caspases 3, 8, and 9 and NF-kappaB as components of p53-targeting mechanisms in embryos exposed to the teratogen.

Transcription factor NF-κB must be released from cytoplasmic inhibitory molecules (IκBs) inorder to move to the nucleus and to activate its target genes. Little is known about the mechanisms regulating the maintenance of constitutive nuclear NF-κB in some cell-types and of sustained nuclear NF-κB activity after stimulation. Increased turnover has been implicated in the regulation of constitutive NF-κB activity in mature B cells. We therefore compared the turnover of IκBα and IκBβ in mature B cells and HeLa cells. Both proteins display a high turnover in B cells although IκBβ is considerably more stable than IκBα. The half-life of both inhibitors is increased in HeLa cells. In contrast, all other NF-κB/IκB molecules tested are relatively stable in both cell-types. The elevated turnover of endogenous IκBα in Namalwa cells is inhibited by a proteasome inhibitor and thus seems to be driven by the same degradation machinery as the slower turnover in non-B cells. Furthermore, we investigated the processes involved in persistent activation of NF-κB. TNF-α signaling leads to a rapid depletion of cellular IκBβ pools. IκBα is efficiently resynthesized whereas IκBβ levels stay low for a prolonged time. NF-κB binding activity can be detected for several hours after stimulation. We found that removal of the TNF-α containing medium causes a rapid decrease in nuclear NF-κB. A phosphoform of newly synthesized IκBα is visible when degradation by the proteasome is inhibited and new IκBα displays the same properties regarding phosphorylation and degradation in response to a second inducer. There is no significant difference in the turnover of pre- and post-inductive IκBα. These observations suggest that resynthesis of IκBα and removal of the stimulus are obligatory steps for the inactivation of nuclear NF-κB.

NF-kappaB was shown previously to regulate apoptotic cell death processes in various experimental systems. However, its role in controlling teratogen-induced cell death has not been established yet. Therefore, the objective of the present study was to explore the involvement of the p65 subunit of NF-kappaB in the response of mouse embryonic fibroblasts (MEFs) to heat shock, using p65 knockout (p65-/-) cells. Indeed, we found p65-/- MEFs to be more susceptible to the exposure to heat shock, as compared with wild-type (WT) MEFs, as they demonstrated a more prominent decrease in cell survival and proliferation as well as the appearance of cells undergoing apoptotic cell death. These heat-shock-induced effects were preceded by a decrease in p65 expression in WT cells, which was accompanied by a decrease in IkappaBalpha expression in WT MEFs, while disappearing completely in p65-/- MEFs and accordingly, by an increase in p-IkappaBalpha expression in both cell lines, which was found to be more prominent in p65-/- MEFs. Interestingly, the heat shock-induced decrease in p65 expression was accompanied by an increase in HSP70 expression in both cell lines. However, it was again found to be more prominent in p65-/- MEFs. Taken together, our results suggest a protective role for the p65 subunit of NF-kappaB in mechanisms underlying the response of embryonic cells to heat shock.

observed previously that tumor necrosis factor alpha (TNF alpha)-knockout embryos are more sensitive to a cyclophosphamide (CP)-induced teratogenic insult than their TNF alpha-positive counterparts, implicating molecules acting in TNFot-activated antiapoptotic pathways in the mechanisms underlying this phenomenon. The main goal of this study was to assess whether the transcription factor nuclear factor kappa B (NF-kappa B) may be 1 of those molecules. Such a choice is based by evidence demonstrating TNF alpha as a powerful activator of NF-kappa B and a key role of the transcription factor in the most effective TNF alpha-activated antiapoptotic cascade. Also, the expression pattern of active caspases 3. 8, and 9 was researched to assess the sensitivity of TNF alpha(+/+) and TNF alpha(-/-) embryos to CP-induced apoptotic stimuli. METHODS: TNF alpha-knockout mice were exposed to CP on day 12 of pregnancy, with or without an NF-kappa B inhibitor, pyrrolidine dithiocarbamate (PDTC) and sacrificed on day 18 of pregnancy to evaluate the CP-induced teratogenic effect. Embryos harvested 24 or 48 hr after the CP treatment were used to evaluate NF-kappa B DNA-binding and activity of caspases 3, 8, and 9. RESULTS: PDTC potentiated the CP-induced teratogenic effect and augmented the CP-induced suppression of NF-kappa B DNA-binding. These effects were more prominent in TNF alpha(-/-) than TNF alpha(+/+) embryos. CP-induced caspase activation was found to be similar in TNF alpha(-/-) and TNFa(+/+) embryos at 24 hr after treatment. At 48 hr, TNF alpha(-/-) embryos exhibited higher levels of active caspases 8 and 9 than their TNF alpha-positive counterparts. CONCLUSIONS: The results of our study allow us to hypothesize that NF-kappa B may be a component of mechanisms underlying differential sensitivity of TNF alpha(-/-) and TNF alpha(+/+) mice to CP-induced teratogenic insult.

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-κB and IκBα 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 IκBα in the response of the embryo and the placenta to teratogenic insults.

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)‐κB DNA‐binding activity in the embryos of immunopotentiated mice exposed to cyclophosphamide (CP).Methods of study The rate of resorptions and the proportion of malformed fetuses in CP‐treated mice were recorded on day 19 of pregnancy. Activity of caspases was tested in cytoplasmic extracts collected from the embryonic brain 24 hr after CP treatment using appropriate fluorometric kits, whereas NF‐κB DNA‐binding activity was evaluated in nuclear extracts using the electrophoretic mobility shift assay.Results As in our previous studies, immunopotentiated CP‐treated females exhibited a lower rate of resorptions or fetuses with open eyes than their non‐immunopotentiated counterparts. In parallel, we observed that maternal immunopotentiation normalized the CP‐induced activation of the tested caspases as well as the CP‐induced suppression of NF‐κB DNA‐binding activity.Conclusions As caspases act as inducers of apoptosis, and NF‐κB acts in CP‐treated embryos as an apoptosis suppressor, the above results suggest that maternal immunopotentiation might affect embryonic sensitivity to embryopathic stresses via NF‐κB‐ and caspases‐associated pathways.

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. Also, a different pattern of intracellular localization of p65 in WT cells as well as IkappaBalpha and Bax in both cell lines was detected in response to MTX. Altogether, our results implicate the p65 subunit of NF-kappaB to play an important role in the response of embryonic cells to MTX.