Teratogen-induced activation of ERK, JNK, and p38 MAP kinases in early postimplantation murine embryos.

BACKGROUND: Although many teratogens are known to activate apoptotic pathways culminating in abnormal development, little is known about how the embryo transduces a teratogenic exposure into specific responses. Signal reception and transduction are regulated by a number of signal transduction pathways, including the extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the stress-activated protein kinase, p38. METHODS: To analyze the effects of teratogens on MAP kinases, we used whole embryo culture, Western blot analyses, and antibodies recognizing inactive or active MAP kinases, or both. RESULTS: We show that heat shock (HS) induces a rapid, strong, but transient activation of ERK, JNK, and p38 with maximal activation occurring within 30 min of the heat shock. By contrast, cyclophosphamide (CP) and staurosporine (ST) failed to activate ERK or JNK during the time period studied (7. 5 hr). ST and CP did induce a low but reproducible activation of p38 beginning at around 3 hr and 5 hr, respectively, after the initiation of exposure. Previous work has shown that heat shock induces elevated cell death in the embryo, primarily in the developing neuroepithelium, but not in the embryonic heart. Thus, we also compared the activation of these three MAP kinase pathways in heads, hearts, and trunks isolated from day 9 embryos exposed to 43 degrees C for 15 min. The results show that ERK, JNK, and p38 are activated in heads, hearts, and trunks. CONCLUSIONS: Our results show that day 9 embryos do activate MAP kinase signaling pathways in response to teratogenic exposures; however, activation of a particular pathway does not appear to be required for teratogen-induced apoptosis.

Heat shock protein 70 (Hsp70) protects postimplantation murine embryos from the embryolethal effects of hyperthermia.

Previous work has shown that there is a positive correlation between the induction of Hsp70 and its transient nuclear localization and the acquisition and loss of induced thermotolerance in postimplantation rat embryos. To determine whether Hsp70 is sufficient to induce thermotolerance in postimplantation mammalian embryos, we used a transgenic mouse in which the normally strictly inducible Hsp70 is constitutively expressed in the embryo under the control of a beta-actin promoter. Day 8.0 mouse embryos heterozygous for the Hsp70 transgene were not protected from the embryotoxic effects of hyperthermia (43 degrees C); however, homozygous embryos, expressing approximately twice as much Hsp70 as heterozygous embryos, were partially protected (increased embryo viability) from the embryolethal effects of hyperthermia. Although the viability of transgenic embryos was significantly increased compared with that of nontransgenic embryos, this protection did not extend to embryo growth and development. To determine whether the failure to achieve a more robust protection was related to the expression of insufficient Hsp70 in transgenic embryos, we undertook experiments to determine whether the level of Hsp70 correlated with the level of thermotolerance induced by various lengths of a 41 degrees C heat shock. A 41 degrees C, 5-minute heat shock failed to induce Hsp70 or thermotolerance, a 41 degrees C, 15-minute heat shock induced Hsp70 and a significant level of thermotolerance, while a 41 degrees C, 60-minute heat shock induced an even higher level of Hsp70 as well as a higher level of thermotolerance. Quantitation of Hsp70 levels indicated that thermotolerance was associated with levels of Hsp70 of 820 pg/microg embryo protein or greater. Subsequent quantitation of the amount of Hsp70 expressed in homozygous transgenic embryos indicated a level of 577 pg/microg embryo protein, that is, a level below that associated with induced thermotolerance. Overall, results presented indicate that Hsp70 does play a direct role in the induction of thermotolerance in postimplantation mouse embryos; however, the level of thermotolerance is dependent on the level of Hsp70 expressed.

Induction of thermotolerance in early postimplantation rat embryos is associated with increased resistance to hyperthermia-induced apoptosis.

Previously we reported that hyperthermia (43 degrees C) induces cell death in neurulation stage rat embryos as part of the pathogenesis culminating in abnormal growth and development. We now show that hyperthermia-induced cell death occurs by a process termed apoptosis. DNA fragmentation, a hallmark of apoptosis, was noted as early as 2.5 hr after embryos were exposed to 43 degrees C. A smaller but significant increase in DNA fragmentation was also observed in embryos exposed to 42 degrees C, but only at the 5 hr time point. In control embryos, TUNEL-positive apoptotic bodies were consistently observed in the neuroepithelium at the point of neural tube closure and in the optic stalk. In embryos exposed to 43 degrees C, the number of TUNEL-positive apoptotic bodies was significantly increased. Using both gel electrophoresis and TUNEL, we also show that the induction of thermotolerance is associated with a significant reduction in DNA fragmentation. Together our results show that specific programmed cell death and hyperthermia-induced cell death correlate with internucleosomal DNA fragmentation characteristic of apoptosis. Finally, we show that the induction of thermotolerance in rat embryos is associated with a significant reduction in internucleosomal DNA fragmentation and associated apoptosis.

The teratogenicity of N(omega)-nitro-L-ariginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, in rats.

Exposure of gravid rats to the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water or by implanted osmotic minipumps significantly elevates maternal blood pressure, reducing uteroplacental perfusion. Administration by either route causes fetal growth retardation, but oral exposure also causes hind limb reduction malformations. The present study employed both oral and intraperitoneal routes to determine the period of sensitivity to developmental toxicity, dose-response, and possible fetotoxic mechanisms. Hind limb hemorrhage occurred only in litters from dams exposed to oral doses of 1 to 2 mg/mL from gestational days 15 through 17. In contrast to oral exposure, single intraperitoneal injections caused both fore and hind limb reductions at doses of 25 mg/kg and above administered on gestational day 16 and later. Many other exposures that reduce uteroplacental perfusion have been associated with vascular disruptive dysmorphogenesis. These exposures include phenytoin, calcium channel inhibitors, cocaine, and uterine vascular clamping. Limb hemorrhage induced by these exposures is usually limited to distal structures, typically phalanges, and the incidence of affected fetuses rarely exceeds 50%. By contrast, hemorrhage caused by L-NAME frequently involves entire limbs, extending into adjacent flank in severe instances, and 100% of fetuses from treated dams may be affected. The basis of this difference and the differing defect patterns associated with the various routes of exposure are unclear, but the generation of reactive oxygen species during resumption of normal perfusion may play a role in this vascular disruption.