Hasida Orenstein

TNF‐α Protects Embryos Exposed to Developmental Toxicants

BACKGROUND:  Tumor necrosis factor α (ΤNF‐α) 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‐α further, a comparative study was performed in TNF‐α knockout and TNF‐α positive mice, exposed to a reference teratogen, cyclophosphamide (CP).

Apoptosis in the Uterus of Mice with Pregnancy Loss

PROBLEM: The mechanisms mediating pregnancy loss induced by various agents are far from being understood. Thus, we investigated the possible involvement of one such mechanism, the apoptotic process, in pregnancy loss induced by lipopolysaccharide (LPS) or cyclophosphamide (CP) as well as the associated changes in the apoptosis‐regulating gene products p53 and bcl‐2.

Immunostimulation increases the resistance of mouse embryos to the teratogenic effect of diabetes mellitus

Summary The present work was aimed to assess the possible effect of stimulation of the maternal immune system on the teratogenic potential of diabetes mellitus. ICR female mice were immunized with splenocytes of male rats 3 weeks before the beginning of mating and were injected with 240 mg/kg streptozocin (STZ) 10 days after immunization. Females with blood glucose levels over 27.8 mmol/l and HbA1 c levels over 6 standard deviations (SD) above the mean of intact animals were used for teratological studies. The rate of malformed fetuses, resorptions and fetal weights were evaluated for animals killed on day 19 of pregnancy using routine teratological methods. Also, phenotyping of spleen cells of these females was performed by fluorescein activated cell sorter analysis. Two main effects possibly due to immunostimulation of ICR females were observed: 1) immunostimulated females had significantly fewer litters with malformed fetuses than non-immunized females: only 4 litters out of 22 (18 %) compared to 10 out of 16 (63 %). Correspondingly, the incidence of malformed fetuses was also decreased: 2.1 compared to 8.9 %; 2) a significant increase in the pregnancy rate in immunized diabetic ICR mice: 69 % as compared to 44 % in non-immunized diabetic females. Also, immunostimulation resulted in a visible increase in spleen cellularity and a certain increase in the number of cells with mature T-cell and macrophage surface markers. These results strongly suggest that immunostimulation increases the tolerance of ICR females to the teratogenic effect of STZ-induced diabetes. [Diabetologia (1997) 40: 635–640]

NF-κB DNA-binding activity in embryos responding to a teratogen, cyclophosphamide

BackgroundThe Rel/NF-κB 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-κB subunits, RelA, has been shown to be crucial for normal embryonic development, in which it functions in the embryonic liver as a protector against TNFα-induced physiological apoptosis. This study assesses whether NF-κB may be involved in the embryos response to teratogens. Fot this, we evaluated how NF-KappaB DNA binding activity in embryonic organs demonstraiting 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).ResultsThe 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-κB 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-κB 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-κB DNA-binding activity in these organs.ConclusionThe results of this study demonstrate that suppression of NF-κB 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-κB DNA-binding activity in the embryonic tissues in an organ type- and dose-dependent fashion.

TNF-α expression in embryos exposed to a teratogen

PROBLEM: The role of tumor necrosis factor (TNF)‐α produced by embryonic cells in normal and abnormal development is poorly understood. To assess to what extent TNF‐α may be involved in the process of induced dysmorphogenesis, the expression of TNF‐α and TNF‐α receptor (TNFRI) mRNA as well as TNF‐α protein was evaluated in embryos responding to a cyclophosphamide (CP)‐induced teratogenic insult. The effect of maternal immunostimulation increasing the embryos tolerance to CP on TNF‐α expression was also investigated.

Expression of Tumor Necrosis Factor-α in the Pregnant Uterus of Diabetic Mice: Effect of Maternal Immunopotentiation

PROBLEM: Tumor necrosis factor (TNF)‐α mRNA and protein are expressed in the pregnant uterus of streptozotocin‐induced diabetic mice at various stages of pregnancy. We intend to characterize their pattern and to evaluate whether the potentiation of the maternal immune system alters the pattern of the expression of the cytokine. 
METHOD OF STUDY: Diabetes was induced in ICR mice by streptozotocin injection. To modulate maternal immune responses, ICR mice were injected intrauterine with rat splenocytes 3 weeks before mating. The expression of TNF‐α mRNA and protein was evaluated by in situ hybridization and immunohistochemistry techniques. 
RESULTS. The population of diabetic mice used in this study demonstrated a reduction in pregnancy rate and an increased number of litters with severely malformed fetuses. It has been observed that these disturbances are associated with a clear increase in TNF‐α mRNA and protein expression in the uterus of these mice. Maternal immunopotentiation, while improving reproductive performance of these diabetic mice, was found to be accompanied by a reduced expression of TNF‐α, both at the mRNA and protein level. 
CONCLUSIONS. The results of the present study suggest a possible involvement of TNF‐α in mechanisms underlying diabetes‐associated dismorphogenesis. Normalization of TNF‐α expression by maternal immunopotentiation might represent a mechanism mediating its protective effect against diabetes‐induced embryotoxic insult.

TNF-α acts to prevent occurrence of malformed fetuses in diabetic mice

Aims/hypothesisActivation of apoptosis in embryos is thought to be a key event in the pathogenesis of diabetes-induced embryopathies such as early embryonic death and inborn structural anomalies. TNF-α can activate apoptotic and anti-apoptotic signalling cascades, indicating its ability to contribute to and counteract diabetes-induced maldevelopment. To investigate how TNF-α regulates the response of embryos to diabetes-induced embryopathic stress, we used streptozotocin-induced diabetic TNF-α knockout mice.MaterialsTo evaluate the reproductive performance, mated diabetic female mice were examined on days 4 and 8 of pregnancy for the presence of blastocysts or embryos in uterine horns. To evaluate the teratogenic effect, the female mice were killed on day 18 of pregnancy and fetuses were examined for gross external anomalies. In addition, apoptotic nuclei were localised by the TUNEL assay and DNA-binding activity of the transcription factor NF-κB was evaluated by electrophoretic mobility shift assay in 10- and 11-day-old embryos respectively.ResultsSeverely diabetic TNF-α+/+ female mice had a much greater decrease in pregnancy rate but a lower incidence of malformed fetuses in litters than severely diabetic TNF-α−/− female mice. Also, the intensity of excessive apoptosis was higher, but the amount of active NF-κB complexes was lower in malformed TNF-α−/− embryos than in TNF-α+/+ embryos.Conclusions/interpretationTNF-α contributes to death of peri-implantation embryos and possibly protects postimplantation embryos exposed to diabetes-induced teratogenic stimuli via activation of NF-κB-mediated anti-apoptotic signalling. It seems that TNF-α prevents the birth of malformed offspring in severely diabetic mice.

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.

TGFβ2 in embryos with inborn anomalies : Effect of maternal immunopotentiation

PROBLEM: TGFβs are among the main immunoregulatory molecules contributing to successful embryonic development. Besides, our and other studies revealed that maternal immunopotentiation has a potential to increase the resistance of the embryo to the teratogenic insult. This work was designed to evaluate: (1) whether the formation of teratogen‐induced anomalies is accompanied by an altered pattern of TGFβ2 expression in embryonic cells and (2) whether maternal immunopotentiation modifies the pattern of TGFβ2 expression in embryos responding to the teratogenic insult.
 METHOD OF STUDY: Experiments were performed in embryos of ICR mice exposed to 15 and 40 mg/kg of a reference teratogen, cyclophosphamide (CP) on day 12 of gestation. A group of mice was immunopotentiated with xenogeneic rat splenocytes 21 hr before the beginning of mating. Embryos were examined for the occurrence of gross structural anomalies 24 and 72 hr after CP treatment. Then, immunohistohemistry and in situ hybrydization assays were used to evaluate the expression of TGFβ2 protein and mRNA in the brain, face, limbs and liver of these embryos.
 RESULTS: No external anomalies were observed in embryos examined 24 hr after CP treatment. Embryos examined 72 hr after CP treatment at 40mg/kg exhibited agnathia, micrognathia, kinky tail, phocomelia, but no signs of dismorphogenesis were observed in the liver at the organ level. A significant increase in the expression of TGFβ2 mRNA was observed in cells, residing in the brain, face and limbs but not in the liver of CP‐exposed embryos tested 24 hr after CP injection in both doses. The level of TGFβ2 protein in these embryos did not differ from that of controls. In embryos tested 72 hr after CP injection in the high dose both TGFβ2 protein and mRNA expression were found to be elevated. Maternal immunopotentiation while enhancing the embryos resistance to CP practically abolished an elevated expression of the TGFβ2 mRNA detected in tested organ structures of embryos of non‐immunopotentiated CP treated mice 24 hr after CP injection in both the low and the high doses. Also, a significant decrease in the level of TGFβ2 mRNA expression was observed in embryos of immunopotentiated mice examined 72 hr after CP treatment.
 CONCLUSIONS: The results of this work show a possible involvement of TGFβ2 in the formation of teratogen‐induced structural anomalies and suggest that the stimulation of the maternal immune system may realize its protective effect by normalizing the level of TGFβ2 expression in teratogen‐targeted embryonic structures.

Bax-associated mechanisms underlying the response of embryonic cells to methotrexate.

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-G(1) 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 IkappaBalpha-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.