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. 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.

Mechanisms of the embryo's response to embryopathic stressors: a focus on p53.

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.

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.

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.

TNFalpha in the pathogenesis of diabetes-induced embryopathies: functions and targets.

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.

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.

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.

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.

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.

Characterization of a novel murine retrovirus mixture that facilitates hematopoiesis.

A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med. 3:37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1(+) Lin(-) hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments. Emv1 was expressed in the spleens of multiparous mice but not in those of virgin mice, and Bxv1Emv1-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.

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.