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Philip E Mirkes - One of the best experts on this subject based on the ideXlab platform.
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inducible 70 kda heat shock proteins protect embryos from Teratogen induced exencephaly analysis using hspa1a a1b knockout mice
Birth Defects Research Part A-clinical and Molecular Teratology, 2009Co-Authors: Philip E Mirkes, Marianne Barrier, David J DixAbstract:BACKGROUND: It is well known that a variety of Teratogens induce neural tube defects in animals; however, less is known about proteins that play a role in protecting embryos from Teratogen-induced neural tube defects. Previously, our laboratory has shown that embryos overexpressing the 70-Da heat shock proteins (HSPs) Hspa1a and Hspa1b were partially protected from the deleterious effects of exposure to hyperthermia in vitro. METHODS: In the present studies, we have used a transgenic mouse in which both of the stress-inducible HSPs Hspa1a and Hspa1b were deleted by homologous recombination. Time-mated Hspa1a/a1b-/- (KO) and wildtype (WT) mice were exposed to hyperthermia in vivo on gestational day 8.5. RESULTS: Results show that 52% of the gestational day 15 fetuses from KO litters were exencephalic, whereas only 20% of WT fetuses were affected. In addition, 6% of treated KO fetuses also exhibited eye defects (microphthalmia and anopthalmia), defects not observed in WT fetuses exposed to hyperthermia. Lysotracker red staining and caspase-3 enzyme activity were examined within 10 hours after exposure to hyperthermia, and significantly greater levels of apoptosis and enzyme activity were observed in the KO embryos compared with WT embryos. CONCLUSIONS: These results show that embryos lacking the Hspa1a and Hspa1b genes are significantly more sensitive to hyperthermia-induced neural tube and eye defects, and this increased sensitivity is correlated with increased amounts of apoptosis. Thus, these results also suggest that Hspa1a and Hspa1b play an important role in protecting embryos from hyperthermia-induced congenital defects, possibly by reducing hyperthermia-induced apoptosis. Birth Defects Research (Part A) 2009. © 2009 Wiley-Liss, Inc.
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Teratogen-induced activation of p53 in early postimplantation mouse embryos.
Toxicological Sciences, 2006Co-Authors: Hiromi Hosako, Sally A. Little, Marianne Barrier, Philip E MirkesAbstract:Hyperthermia (HS) and 4-hydroperoxycyclophosphamide (4CP) activate the mitochondrial apoptotic pathway in day 9 mouse embryos. Previous microarray analyses Microarray analyses revealed that several p53 target genes are upregulated after exposure to HS or 4CP, suggesting a role for p53 in Teratogen-induced apoptosis. To explore the role of p53, we assessed the activation of p53 in day 9 mouse embryos exposed to HS or 4CP in vitro. Both Teratogens induced the accumulation of p53 and phosphorylation of p53 at ser-15, two hallmarks of p53 activation. HS and 4CP also induced an increase in Noxa and Puma mRNAs, transcripts of two known proapoptotic p53 target genes; however, these two Teratogens did not induce significant increases in NOXA and PUMA proteins, suggesting that p53 does not activate the mitochondrial apoptotic pathway by transcriptionally upregulating the expression of NOXA and PUMA proteins. HS and 4CP also induced the expression of p21 mRNA and protein, suggesting a role for p53 in Teratogen-induced cell cycle arrest. Previously, we also showed that HS and 4CP activate the apoptotic pathway in the embryo proper (head and trunk) but not in the heart. We now show that HS and 4CP induce a robust activation of p53 in the embryo proper but an attenuated induction in the heart. HS and 4CP induce the expression of p21 protein in majority of the cells in the embryo; however, expression of NOXA and PUMA proteins were not significantly induced in heads, hearts, or trunks of day 9 embryos. Overall, our results suggest that p53 may play a transcription-dependent role in Teratogen-induced cell cycle arrest but a transcription-independent role in Teratogen-induced apoptosis in day 9 mouse embryos exposed to HS or 4CP.
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Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos
Cell Biology and Toxicology, 2003Co-Authors: S.a. Little, Philip E MirkesAbstract:Background : Previous work has shown that Teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to Teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by Teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in Teratogen-induced cell death is unknown. Methods : Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between Teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. Results : Western blot analyses show that these three Teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three Teratogens. Conclusions : HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that Teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that Teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to Teratogen-induced cell death.
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Teratogen-induced activation of the mitochondrial apoptotic pathway in the yolk sac of day 9 mouse embryos.
Birth defects research. Part A Clinical and molecular teratology, 2003Co-Authors: Donna Soleman, Leanne Cornel, Sally A. Little, Philip E MirkesAbstract:BACKGROUND Using vital dyes, we have previously shown that while hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death within specific tissues (e.g., neuroepithelium) of day 9 mouse embryos, cells of the heart are resistant to the cell death-inducing potential of these Teratogens. Subsequent work has shown that Teratogen-induced cell death is associated with activation of the mitochondrial apoptotic pathway, i.e., release of cytochrome c from mitochondria, activation/cleavage of procaspase-9, -3, and -2, inactivation of poly(ADP-ribose) polymerase, and internucleosomal fragmentation of DNA, whereas resistance to Teratogen-induced cell death in the heart is associated with a failure to activate this pathway. Teratogen-induced activation of the mitochondrial apoptotic pathway is initiated between 2.5 and 5 hr after Teratogens are added to the culture medium. Because both the heart and the surrounding yolk sac are essential to successful development of mouse embryos during early postimplantation mouse development, we hypothesized that cells of the yolk sac are also resistant to Teratogen-induced cell death. METHODS To test our hypothesis, we cultured day 8.5 mouse conceptuses (embryo plus yolk sac) in whole embryo culture. On the morning of day 9, conceptuses were exposed to HS (43°C for 15 min and then returned to 37°C), 4CP (40 μM, 5–10 hr), or ST (0.5 μM, 5–10 hr). At 5 and 10 hr after addition of Teratogen, conceptuses were removed from culture and dissected into embryo and yolk sac. Activation of the mitochondrial apoptotic pathway was then assessed separately in embryos and yolk sacs using Western blot analysis to detect activation of procaspase-9, -3, and -2, enzyme assays to measure caspase-3–like activity, and immunohistochemistry to detect caspase-3 activation/cleavage in yolk sac cells. RESULTS Although Western blot analysis revealed that procaspase-9, -3, and -2 were activated/cleaved in the embryo as early as the 5-hr time point, activation/cleavage of these caspases could not be detected in the yolk sac at either the 5- or 10-hr time point. Using an enzyme assay, we determined that caspase-3–like activity in the yolk sac was induced 1.7-fold by HS, 4.4-fold by 4CP, and 3.3-fold by ST. This compares to the embryo in which caspase-3–like activity was induced 45-fold by HS, 26-fold by 4CP, and 45-fold by ST. Using an antibody specific for the active p17 subunit of caspase-3 and immunohistochemistry, we were able to detect a small number of yolk sac cells showing caspase-3 activation. Thus, the low-level induction of caspase-3–like activity in the yolk sac is in part related to activation/cleavage of procaspase-3. CONCLUSIONS Results presented indicate that cells of the extraembryonic yolk sac, like cells of the embryonic heart, are substantially more resistant to Teratogen-induced activation of the mitochondrial apoptotic pathway and subsequent apoptosis compared to other embryonic tissues, particularly cells of the neuroepithelium. Birth Defects Research (Part A) 67:98–107, 2003. © 2003 Wiley-Liss, Inc.
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Teratogen-Induced Activation of Caspase-9 and the Mitochondrial Apoptotic Pathway in Early Postimplantation Mouse Embryos☆
Toxicology and Applied Pharmacology, 2002Co-Authors: Sally A. Little, Philip E MirkesAbstract:Previously we showed that Teratogen-induced cell death in mouse embryos is apoptotic in nature, i.e., involves the release of cytochrome c from mitochondria and the subsequent activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation. Herein we show that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine also activate caspase-9, the apical caspase in the mitochondrial apoptotic pathway. Activation of procaspase-9 is associated with the cleavage of this proenzyme and the generation of two forms of the large subunit, primarily a 39-kDa subunit (p39) but also a lesser amount of a 37-kDa subunit (p37). We also present data that support the idea that the Teratogen-induced formation of the p37 subunit in vivo occurs by the cytochrome c-mediated processing of procaspase-9, whereas the p39 subunit is formed by an amplification loop involving caspase-3. We also previously showed that the release of cytochrome c, activation of caspase-3, cleavage of PARP, and DNA fragmentation are blocked in cells of the developing heart, which are resistant to Teratogen-induced cell death. We now show that this block in the mitochondrial apoptotic pathway in heart cells extends to the activation of procaspase-9. Thus, our cumulative data indicate that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine induce cell death in Day 9 mouse embryos by activating the mitochondrial apoptotic pathway. In addition, our data suggest that cells of the Day 9 mouse embryo that are resistant to Teratogen-induced cell death possess multiple mechanisms for inhibiting the mitochondrial apoptotic pathway after a Teratogenic exposure.
Robert P. Erickson - One of the best experts on this subject based on the ideXlab platform.
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confirmation of the role of n acetyltransferase 2 in Teratogen induced cleft palate using transgenics and knockouts
Molecular Reproduction and Development, 2008Co-Authors: Robert P. Erickson, Diana K. Acuña, Wen Cao, Diana Strnatka, Robert J Hunter, Binh Chau, Larissa Wakefield, Edith Sim, Charlene A McqueenAbstract:Previous work on Dilantin- and hydrocortisone-induced cleft palate and cleft lip with or without cleft palate using congenics for the N-acetyltransferase loci (Nat1 and Nat2 are closely linked) and recombinant inbred lines implicated the Nat1,2 region in susceptibility to Teratogen-induced orofacial clefting. Since Nat1 does not differ between the two strains, Nat2 appeared to be responsible. We have now tested this conclusion using transgenics and knockouts. Transgenics for human NAT1 (equivalent to mouse Nat2) and knockouts for Nat2 were tested for susceptibility to Dilantin, hydrocortisone, and 6-aminonicotinamide-induced orofacial clefting. We found that Nat2 greatly influences Teratogen-induced orofacial clefting on the A/J background but not on the C57BL/6J background. The magnitude and direction of the effects depended on which Teratogen was used. The Nat2 knockout did not make C57BL/6J susceptible or A/J (already with very low activity) more susceptible but significantly decreased sporadic clefting in the A/J strain. We conclude that only the A/J strain, with several loci affecting orofacial clefting, is influenced by Nat2. Mol. Reprod. Dev. 75: 1071–1076, 2007. © 2007 Wiley-Liss, Inc.
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The role of multiple drug resistance proteins in fetal and/or placental protection against Teratogen-induced orofacial clefting.
Molecular Reproduction and Development, 2007Co-Authors: Lesli Ann Rawles, Diana K. Acuña, Robert P. EricksonAbstract:Previous studies have shown a role for multiple drug resistance proteins in protecting the fetus from a limited number of Teratogens. We have expanded the number of proteins and Teratogens examined by comparing the influence of the mdr1a and mdr2 proteins on Teratogen-induced orofacial clefting using their respective knockouts in crosses with the A/J, high susceptibility strain. Western blots identified the presence of mdr1a and possibly mdr2 in the placenta and fetus. The mdr1a knockout, on its unique genetic background showed lower, similar, and higher incidences of clefting compared to A/J for Dilantin, hydrocortisone (HC), and 6-aminonicotinamide (6-AN), respectively. The mdr2 knockout did not affect 6-AN clefting when compared to A/J. In reciprocal crosses, when corrected for increased spontaneous clefting, maternally inherited A/J susceptibility genes predominated over the effects of the maternal absence of mdr1a (with 6-AN). Unlike mdr1a, which had a direct effect in the fetus as shown by genotyping of affected versus unaffected fetuses, an effect of mdr2 in the fetus was not found. The mdr1a knockout was backcrossed to the A/J inbred strain for 11 generations (congenics) to eliminate genetic background effects. Reciprocal crosses showed no maternal effect from the lack of mdr1a, confirming that mdr1a expression in the fetus, rather than the placenta, protects the fetus from Teratogens. Mdr2 seems not to be involved in the protection of the fetus from Teratogens. Mol. Reprod. Dev. 74: 1483–1489, 2007. © 2007 Wiley-Liss, Inc.
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the role of multiple drug resistance proteins in fetal and or placental protection against Teratogen induced orofacial clefting
Molecular Reproduction and Development, 2007Co-Authors: Lesli Ann Rawles, Diana K. Acuña, Robert P. EricksonAbstract:Previous studies have shown a role for multiple drug resistance proteins in protecting the fetus from a limited number of Teratogens. We have expanded the number of proteins and Teratogens examined by comparing the influence of the mdr1a and mdr2 proteins on Teratogen-induced orofacial clefting using their respective knockouts in crosses with the A/J, high susceptibility strain. Western blots identified the presence of mdr1a and possibly mdr2 in the placenta and fetus. The mdr1a knockout, on its unique genetic background showed lower, similar, and higher incidences of clefting compared to A/J for Dilantin, hydrocortisone (HC), and 6-aminonicotinamide (6-AN), respectively. The mdr2 knockout did not affect 6-AN clefting when compared to A/J. In reciprocal crosses, when corrected for increased spontaneous clefting, maternally inherited A/J susceptibility genes predominated over the effects of the maternal absence of mdr1a (with 6-AN). Unlike mdr1a, which had a direct effect in the fetus as shown by genotyping of affected versus unaffected fetuses, an effect of mdr2 in the fetus was not found. The mdr1a knockout was backcrossed to the A/J inbred strain for 11 generations (congenics) to eliminate genetic background effects. Reciprocal crosses showed no maternal effect from the lack of mdr1a, confirming that mdr1a expression in the fetus, rather than the placenta, protects the fetus from Teratogens. Mdr2 seems not to be involved in the protection of the fetus from Teratogens. Mol. Reprod. Dev. 74: 1483–1489, 2007. © 2007 Wiley-Liss, Inc.
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genome scan for Teratogen induced clefting susceptibility loci in the mouse evidence of both allelic and locus heterogeneity distinguishing cleft lip and cleft palate
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Scott R Diehl, Robert P. EricksonAbstract:Nonsyndromic clefting of the lip and palate in humans has a highly complex etiology, with both multiple genetic loci and exposure to Teratogens influencing susceptibility. Previous studies using mouse models have examined only very small portions of the genome. Here we report the findings of a genome-wide search for susceptibility genes for Teratogen-induced clefting in the AXB and BXA set of recombinant inbred mouse strains. We compare results obtained using phenytoin (which induces cleft lip) and 6-aminonicotinamide (which induces cleft palate). We use a new statistical approach based on logistic regression suitable for these categorical data to identify several chromosomal regions as possible locations of clefting susceptibility loci, and we review candidate genes located within each region. Because cleft lip and cleft palate do not frequently co-aggregate in human families and because these structures arise semi-independently during development, these disorders are usually considered to be distinct in etiology. Our data, however, implicate several of the same chromosomal regions for both forms of clefting when Teratogen-induced. Furthermore, different parental strain alleles are usually associated with clefting of the lip versus that of the palate (i.e., allelic heterogeneity). Because several other chromosomal regions are associated with only one form of clefting, locus heterogeneity also appears to be involved. Our findings in this mouse model suggest several priority areas for evaluation in human epidemiological studies.
Vladimir Toder - One of the best experts on this subject based on the ideXlab platform.
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Teratogen induced apoptotic cell death does the apoptotic machinery act as a protector of embryos exposed to Teratogens
Birth Defects Research Part C-embryo Today-reviews, 2005Co-Authors: Arkady Torchinsky, Amos Fein, Vladimir ToderAbstract: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.
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Teratogen‐induced apoptotic cell death: Does the apoptotic machinery act as a protector of embryos exposed to Teratogens?
Birth defects research. Part C Embryo today : reviews, 2005Co-Authors: Arkady Torchinsky, Amos Fein, Vladimir ToderAbstract: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.
Sally A. Little - One of the best experts on this subject based on the ideXlab platform.
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Teratogen-induced activation of p53 in early postimplantation mouse embryos.
Toxicological Sciences, 2006Co-Authors: Hiromi Hosako, Sally A. Little, Marianne Barrier, Philip E MirkesAbstract:Hyperthermia (HS) and 4-hydroperoxycyclophosphamide (4CP) activate the mitochondrial apoptotic pathway in day 9 mouse embryos. Previous microarray analyses Microarray analyses revealed that several p53 target genes are upregulated after exposure to HS or 4CP, suggesting a role for p53 in Teratogen-induced apoptosis. To explore the role of p53, we assessed the activation of p53 in day 9 mouse embryos exposed to HS or 4CP in vitro. Both Teratogens induced the accumulation of p53 and phosphorylation of p53 at ser-15, two hallmarks of p53 activation. HS and 4CP also induced an increase in Noxa and Puma mRNAs, transcripts of two known proapoptotic p53 target genes; however, these two Teratogens did not induce significant increases in NOXA and PUMA proteins, suggesting that p53 does not activate the mitochondrial apoptotic pathway by transcriptionally upregulating the expression of NOXA and PUMA proteins. HS and 4CP also induced the expression of p21 mRNA and protein, suggesting a role for p53 in Teratogen-induced cell cycle arrest. Previously, we also showed that HS and 4CP activate the apoptotic pathway in the embryo proper (head and trunk) but not in the heart. We now show that HS and 4CP induce a robust activation of p53 in the embryo proper but an attenuated induction in the heart. HS and 4CP induce the expression of p21 protein in majority of the cells in the embryo; however, expression of NOXA and PUMA proteins were not significantly induced in heads, hearts, or trunks of day 9 embryos. Overall, our results suggest that p53 may play a transcription-dependent role in Teratogen-induced cell cycle arrest but a transcription-independent role in Teratogen-induced apoptosis in day 9 mouse embryos exposed to HS or 4CP.
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Teratogen-induced activation of the mitochondrial apoptotic pathway in the yolk sac of day 9 mouse embryos.
Birth defects research. Part A Clinical and molecular teratology, 2003Co-Authors: Donna Soleman, Leanne Cornel, Sally A. Little, Philip E MirkesAbstract:BACKGROUND Using vital dyes, we have previously shown that while hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death within specific tissues (e.g., neuroepithelium) of day 9 mouse embryos, cells of the heart are resistant to the cell death-inducing potential of these Teratogens. Subsequent work has shown that Teratogen-induced cell death is associated with activation of the mitochondrial apoptotic pathway, i.e., release of cytochrome c from mitochondria, activation/cleavage of procaspase-9, -3, and -2, inactivation of poly(ADP-ribose) polymerase, and internucleosomal fragmentation of DNA, whereas resistance to Teratogen-induced cell death in the heart is associated with a failure to activate this pathway. Teratogen-induced activation of the mitochondrial apoptotic pathway is initiated between 2.5 and 5 hr after Teratogens are added to the culture medium. Because both the heart and the surrounding yolk sac are essential to successful development of mouse embryos during early postimplantation mouse development, we hypothesized that cells of the yolk sac are also resistant to Teratogen-induced cell death. METHODS To test our hypothesis, we cultured day 8.5 mouse conceptuses (embryo plus yolk sac) in whole embryo culture. On the morning of day 9, conceptuses were exposed to HS (43°C for 15 min and then returned to 37°C), 4CP (40 μM, 5–10 hr), or ST (0.5 μM, 5–10 hr). At 5 and 10 hr after addition of Teratogen, conceptuses were removed from culture and dissected into embryo and yolk sac. Activation of the mitochondrial apoptotic pathway was then assessed separately in embryos and yolk sacs using Western blot analysis to detect activation of procaspase-9, -3, and -2, enzyme assays to measure caspase-3–like activity, and immunohistochemistry to detect caspase-3 activation/cleavage in yolk sac cells. RESULTS Although Western blot analysis revealed that procaspase-9, -3, and -2 were activated/cleaved in the embryo as early as the 5-hr time point, activation/cleavage of these caspases could not be detected in the yolk sac at either the 5- or 10-hr time point. Using an enzyme assay, we determined that caspase-3–like activity in the yolk sac was induced 1.7-fold by HS, 4.4-fold by 4CP, and 3.3-fold by ST. This compares to the embryo in which caspase-3–like activity was induced 45-fold by HS, 26-fold by 4CP, and 45-fold by ST. Using an antibody specific for the active p17 subunit of caspase-3 and immunohistochemistry, we were able to detect a small number of yolk sac cells showing caspase-3 activation. Thus, the low-level induction of caspase-3–like activity in the yolk sac is in part related to activation/cleavage of procaspase-3. CONCLUSIONS Results presented indicate that cells of the extraembryonic yolk sac, like cells of the embryonic heart, are substantially more resistant to Teratogen-induced activation of the mitochondrial apoptotic pathway and subsequent apoptosis compared to other embryonic tissues, particularly cells of the neuroepithelium. Birth Defects Research (Part A) 67:98–107, 2003. © 2003 Wiley-Liss, Inc.
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Teratogen-Induced Activation of Caspase-9 and the Mitochondrial Apoptotic Pathway in Early Postimplantation Mouse Embryos☆
Toxicology and Applied Pharmacology, 2002Co-Authors: Sally A. Little, Philip E MirkesAbstract:Previously we showed that Teratogen-induced cell death in mouse embryos is apoptotic in nature, i.e., involves the release of cytochrome c from mitochondria and the subsequent activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation. Herein we show that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine also activate caspase-9, the apical caspase in the mitochondrial apoptotic pathway. Activation of procaspase-9 is associated with the cleavage of this proenzyme and the generation of two forms of the large subunit, primarily a 39-kDa subunit (p39) but also a lesser amount of a 37-kDa subunit (p37). We also present data that support the idea that the Teratogen-induced formation of the p37 subunit in vivo occurs by the cytochrome c-mediated processing of procaspase-9, whereas the p39 subunit is formed by an amplification loop involving caspase-3. We also previously showed that the release of cytochrome c, activation of caspase-3, cleavage of PARP, and DNA fragmentation are blocked in cells of the developing heart, which are resistant to Teratogen-induced cell death. We now show that this block in the mitochondrial apoptotic pathway in heart cells extends to the activation of procaspase-9. Thus, our cumulative data indicate that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine induce cell death in Day 9 mouse embryos by activating the mitochondrial apoptotic pathway. In addition, our data suggest that cells of the Day 9 mouse embryo that are resistant to Teratogen-induced cell death possess multiple mechanisms for inhibiting the mitochondrial apoptotic pathway after a Teratogenic exposure.
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Teratogen-induced cell death in postimplantation mouse embryos: differential tissue sensitivity and hallmarks of apoptosis.
Cell Death & Differentiation, 1998Co-Authors: Philip E Mirkes, Sally A. LittleAbstract:Teratogen-induced cell death is a common event in the pathogenesis associated with tissues destined to be malformed. Although the importance of this cell death is recognized, little information is available concerning the biochemistry of Teratogen-induced cell death. We show that three Teratogens, hyperthermia, cyclophosphamide and sodium arsenite induce an increase in cell death in day 9.0 mouse embryos with concurrent induction of DNA fragmentation, activation of caspase-3 and the cleavage of poly (ADP-ribose) polymerase (PARP). Teratogen-induced cell death is also selective, i.e., some cells within a tissue die while others survive. In addition, cells within some tissues die when exposed to Teratogens while cells in other tissues are relatively resistant to Teratogen-induced cell death. An example of the latter selectivity is seen in the cells of the developing heart, which are resistant to the cytotoxic potential of many Teratogens. We show that the absence of cell death in the heart is accompanied by the complete lack of DNA fragmentation, activtion of caspase-3 and the cleavage of PARP.
Arkady Torchinsky - One of the best experts on this subject based on the ideXlab platform.
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Teratogen induced apoptotic cell death does the apoptotic machinery act as a protector of embryos exposed to Teratogens
Birth Defects Research Part C-embryo Today-reviews, 2005Co-Authors: Arkady Torchinsky, Amos Fein, Vladimir ToderAbstract: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.
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Teratogen‐induced apoptotic cell death: Does the apoptotic machinery act as a protector of embryos exposed to Teratogens?
Birth defects research. Part C Embryo today : reviews, 2005Co-Authors: Arkady Torchinsky, Amos Fein, Vladimir ToderAbstract: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.
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NF-κB DNA-binding activity in embryos responding to a Teratogen, cyclophosphamide
BMC Developmental Biology, 2002Co-Authors: Arkady Torchinsky, Shoshana Savion, Lucy Lishanski, Orit Wolstein, Jeanne Shepshelovich, Hasida Orenstein, Zeev Zaslavsky, Howard Carp, Alexander Brill, Rivka DiksteinAbstract:Background The 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 embryo's 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).
