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. 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.

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.

Renal apoptosis following carbon dioxide pneumoperitoneum in a rat model.

PURPOSE: Laparoscopically recruited kidneys regain normal function more slowly than laparotomy harvested organs for several possible reasons. We investigated the effects of CO(2) induced pneumoperitoneum on kidney function, as reflected by blood and urine creatinine levels, and its relation with renal cell apoptosis. MATERIALS AND METHODS: CO(2) pneumoperitoneum was established in anesthetized Wistar male rats that were randomly allocated at 6 per group into 1 of 6 groups with an intraperitoneal pressure of 0 (control), 5, 8, 12, 15 or 18 mm Hg. Pressure was maintained for 60 minutes in all groups. Three additional groups were subjected to 30-minute pneumoperitoneum at 0, 12 and 18 mm Hg, respectively. The rats were kept alive for the ensuing 24 hours, after which blood and urine creatinine were analyzed and the abdominal organs were harvested. Various areas of the organs were analyzed for apoptotic cells using the TUNEL method. Cells were randomly counted in 10 eyeshots in 3 sections each using an ocular micrometer. RESULTS: Creatinine levels in blood and urine changed as pressure and pneumoperitoneum duration progressed. Isolated TUNEL positive nuclei were detected in the outer medulla and the cortex of control kidneys. There was a significantly higher number of TUNEL positive nuclei in the cortex and the medulla of all pressurized kidneys (p <0.05), which increased in parallel with increasing intraperitoneal pressure and pneumoperitoneum exposure time. CONCLUSIONS: The CO(2) pneumoperitoneum gradient and its duration affect renal function and induce apoptosis. This could be a mechanism involved in renal delayed graft dysfunction in recipients of laparoscopically harvested kidneys.

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. Embryos of females exposed to 15 mg/kg CP exhibited only a decrease in NF-kappaB-DNA binding and IKKbeta activity at 24 h. However, at 48 h, a more prominent decrease in NF-kappaB activity was observed, accompanied by a decreased expression of p65 and IKKbeta 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-kappaB-DNA binding activity and no alterations in NF-kappaB signaling. These results suggest that the classical NF-kappaB 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-kappaB signaling may culminate in such a final effect as embryonic death.

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. 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.

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.

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. Also, the expression pattern of active caspases 3, 8, and 9 was researched to assess the sensitivity of TNFalpha+/+ and TNFalpha-/- embryos to CP-induced apoptotic stimuli. METHODS: TNFalpha-knockout mice were exposed to CP on day 12 of pregnancy, with or without an NF-kappaB 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-kappaB 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-kappaB DNA-binding. These effects were more prominent in TNFalpha-/- than TNFalpha+/+ embryos. CP-induced caspase activation was found to be similar in TNFalpha-/- and TNFalpha+/+ embryos at 24 hr after treatment. At 48 hr, TNFalpha-/- embryos exhibited higher levels of active caspases 8 and 9 than their TNFalpha-positive counterparts. CONCLUSIONS: The results of our study allow us to hypothesize that NF-kappaB may be a component of mechanisms underlying differential sensitivity of TNFalpha-/- and TNFalpha+/+ mice to CP-induced teratogenic insult.

Recycling to the plasma membrane is delayed in EHD1 knockout mice.

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.

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. 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.

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). 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-kappaB 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-kappaB DNA-binding activity. CONCLUSIONS: As caspases act as inducers of apoptosis, and NF-kappaB 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-kappaB- and caspases-associated pathways.

The exon 8-containing prosaposin gene splice variant is dispensable for mouse development, lysosomal function, and secretion.

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids.

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. To address this issue, this study was designed to test: 1) whether diabetes-induced fetal growth retardation is preceded by the modulation of NF-kappaB activity in embryos at the late stage of organogenesis and 2) whether apoptosis is altered in these embryos. METHODS: The embryos and placentas of streptozotocin-induced diabetic mice collected on days 13 and 15 of pregnancy were used to evaluate the expression of NF-kappaB, IkappaBalpha and phosphorylated (p)-IkappaBalpha proteins by Western blot analysis and NF-kappaB DNA binding by an ELISA-based method. The detection of apoptotic cells was performed by the TUNEL assay and the expression of a proapoptotic protein Bax was evaluated by the Western blot. RESULTS: The embryos of diabetic mice were significantly growth retarded, whereas the placental weight did not differ in diabetic or control females. Levels of NF-kappaB and p-IkappaBalpha proteins as well as the amount of NF-kappaB DNA binding was lower in embryos of diabetic mice as compared to those in controls. However, neither excessive apoptosis nor an increased Bax expression was found in growth-retarded embryos and their placentas. CONCLUSION: The study herein revealed that diabetes-induced fetal growth retardation is associated with the suppression of NF-kappaB activity in embryos, which seems to be realized at the level of IkappaB degradation.

Teratogen-induced apoptotic cell death: does the apoptotic machinery act as a protector of embryos exposed to teratogens?

Considerable evidence has been collected demonstrating that many teratogens induce apoptotic cell death in embryonic structures that turn out to be malformed in fetuses and newborns. Apoptosis is a genetically regulated process that is realized by the activation of death and pro-survival signaling cascades, and the interplay between these cascades determines whether the cell exposed to apoptotic stimuli dies or survives. Therefore, there is intense interest in understanding how the apoptotic machinery functions in embryos exposed to teratogens. However, the interpretation of the results obtained remains problematic. The main problem is that excessive embryonic cell death, regardless of its nature, if uncompensated for, ultimately leads to maldevelopment or embryonic death. Therefore, we can easily interpret results when the intensity of teratogen-induced cell death and the severity or incidence of teratogen-induced anomalies directly correlate with each other. However, when teratogen-induced cell death is not followed by the formation of anomalies, a usual explanation is that teratogen-induced apoptotic cell death contributes to the renewal of teratogen-targeted cell populations by promoting the removal of injured cells. It is clear that such an explanation leaves vague the role of the anti-apoptotic signaling mechanism (and, hence, the apoptotic machinery as a whole) with respect to protecting the embryo against teratogenic stress. In this review, we summarize the data from studies addressing the function of the apoptotic machinery in embryos exposed to teratogens, and then we discuss approaches to interpreting the results of these studies. We hypothesize that activation of a proapoptotic signaling in teratogen-targeted cell populations is a necessary condition for an anti-apoptotic signaling that counteracts the process of maldevelopment to be activated. If such a scenario is true, we need to modify our approaches to choosing molecular targets for studies addressing this topic.

Conservation of expression and alternative splicing in the prosaposin gene.

Prosaposin is the precursor of four lysosomal activator molecules known as saposins A, B, C and D. It is also secreted and was proposed to be a neurotrophic factor. The neurotrophic function was attributed to the amino terminus of saposin C. In man, mouse and rat prosaposin is transcribed to two major isoforms differing in the inclusion of 9 bps of exon 8 within the saposin B domain. In the present study, we show that there is evolutionary conservation of the prosaposin structure and alternative splicing in chick and zebrafish as well. Moreover, there is conservation in prosaposin expression as tested immunohistochemically in the mouse and chick developing brain. We developed a sensitive assay to quantitate the prosaposin alternatively spliced forms. Our results indicate that, in mouse brain, skeletal and cardiac muscle the exon 8-containing RNA is most abundant, while it is almost absent from visceral and smooth muscle-containing organs. We observed temporal and differential expression of the alternatively spliced prosaposin mRNAs in mouse and chick brain as well as during development. The elevation in the abundance of exon 8-containing prosaposin RNA during mouse and chick brain development may suggest a role for the exon 8-containing prosaposin form in this process.

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.

In Vitro Growth of Rat Blastocyst under the Effect of Azathioprine: (rat/blastocyst/azathioprine/in vitro).

Rat blastocysts were isolated from the uterus on the 5th day after fertilization and set in culture. The effect of azathioprine (Imuran) on the blastocyst's development and on the early trophoblastic differentiation in vitro was investigated. Azathioprine, added to the medium of the blastocyst culture at various concentrations, dose dependently arrested development and had definite cytotoxic effect. In order to study the mechanism of action, a minimal dose of 5 µg/ml, which allowed the survival of about 60% of the blastocysts, was added to the medium after 48 hr of culturing. Under the effect of the substance the area of the spreading blastocyst cells was significantly restricted. It was found, by autoradiographic methods, that the azathioprine affects the development by restraining DNA synthesis in the throphoblastic cells. Concomitantly RNA synthesis was inhibited and protein synthesis was reduced. The observations indicate, that the impairment of the in vitro differentiation of the blastocysts can be a result of the intracellular inhibitory action of the substance.