Toxicological Sciences 69, 183-190 (2002)
Copyright © 2002 by the Society of Toxicology
REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY |
HSP90
, HSP90ß, and p53 Expression following in Vitro Hyperthermia Exposure in Gestation Day 10 Rat Embryos
,
,,2
* Pathology Associates, a Charles River Company, Frederick, Maryland 21701;
National Center for Environmental Assessment, Office of Research and Development, U. S. Environmental Protection Agency, Washington, D.C. 20460; and
Health Sciences Branch, Life Sciences Division, Office of Science and Technology, Center for Devices and Radiological Health, U.S. Food and Drug Administration, Rockville, Maryland 20857
Received November 29, 2001; accepted May 8, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The studies presented here are aimed at understanding the expression of p53, HSP90
, and HSP90ß in gestation day (GD) 10 CD rat embryos. GD 10 rat embryos were exposed in vitro to 37°C or 42°C for 15 min, then cultured at 37°C for 0.5, 1, 3, or 5 h. Immunohistochemistry was performed on formalin-fixed, paraffin embedded, sectioned embryos for p53, HSP90, or HSP90ß expression. p53 expression was minimal in control embryos but was induced with heat exposure. Maximum expression of p53 was observed in rostral tissues, e.g., the optic vesicle, rostral neuroepithelium, and mature (rostral) somites 3 and 5 h after heat exposure. Expression of p53 in the caudal region, such as in mid and caudal neuroepithelium, immature (caudal) somites, and presomitic mesoderm, was moderate compared to rostral areas. No p53 expression was observed in the heart under any condition. The rostral-caudal gradient of p53 expression was not observed for HSP90 expression. HSP90 was induced in heat-exposed embryos beginning at 1 h, predominantly in neural tube and optic vesicle. Moderate but increased expression was observed in the somites of heat-exposed embryos at 3 and 5 h. Expression of p53 was primarily nuclear while HSP90 expression was mostly cytoplasmic. No clear association was observed between heat-induced HSP90 and p53 expression. HSP90ß was expressed extensively in control and heat-exposed embryos. Results indicate that heat induces p53 and HSP90 expression, but not HSP90ß expression, and that HSP90 induction is not likely to be involved in p53 regulation in mammalian embryos. Key Words: hyperthermia; HSP90; p53; developmental toxicity; rat embryo culture.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hyperthermia is known to alter development in humans as well as animals, resulting in a number of defects such as anencephaly/exencephaly, encephalocoele, microencephaly, microphthalmia, talipes, arthrogryposis, and defects of the abdominal wall and limb (reviewed by Graham et al., 1998
). The timing of the exposure during gestation is critical, and studies in our laboratory have shown that heat exposure of GD 10 rat embryos to 42°C for 15–20 min either in vivo or in vitro resulted in abnormal somite segmentation and axial skeletal malformations (Cuff et al., 1993; Kimmel et al., 1993a,b). The mechanism of these heat-induced malformations is not clear. Studies at the cellular level have shown that heat shock causes increased cell death or apoptosis, a decrease in mitotic figures in the neural epithelium and somites (Breen et al., 1999; Mirkes, 1985; Walsh et al., 1993), and a disruption of neural and vascular basement membranes (Edwards, 1971; Nilsen et al., 1984).
It is known that several heat shock proteins (HSPs) such as HSP90, HSP70, HSP47, and HSP20 are induced by heat in the embryo (reviewed by Graham et al., 1998). HSPs are classically thought to be cellular protective agents in living organisms (Craig et al., 1994), and a few studies have suggested that HSPs might play a protective role in heat-induced teratogenesis (Kapron-Bras and Hales, 1991; Mirkes, 1987, 1997; Walsh et al., 1987). HSP90 is a molecular chaperone that binds with and/or forms multiprotein complexes with substrate proteins such as protein kinases and transcription factors, thus regulating their function (Richter and Buchner, 2001). Several studies have shown that HSP90 exists in two isoforms, HSP90 and ß, which play an important role in the regulation of an ever-increasing list of transcription factors and protein kinases (Moore et al., 1989; Pratt, 1998; Rebbe et al., 1989); however, their role in heat-induced teratogenesis is not clear. HSP90 has been shown to be heat-inducible in the zebrafish embryo while HSP90ß is not (Krone and Sass, 1994), and HSP90 is known to associate with b-helix-loop-helix transcription factors such as myoD that are crucial in development (Sass and Krone, 1997).
Environmental stress stimulates tumor suppressor protein p53 accumulation and apoptosis induction in a dose-dependent manner in mouse embryos (Bolaris et al., 2001; Wubah et al., 1996). Studies in different cancer cell lines have shown the association of HSP70 and 90 proteins with mutant and wild type tumor suppressor protein p53 (Blagosklonny et al., 1996; Dasgupta and Momand, 1997; Ohnishi et al., 1995; Selkirk et al., 1994; Whitesell et al., 1998). Wild-type p53 has a short half-life and degrades rapidly when the temperature is above 41°C, losing its conformation and functional capacity (Hainaut et al., 1995), showing that p53 is sensitive to heat and that HSPs induced by heat might influence p53 induction. Therefore, our hypothesis for the current study was that p53, which plays a pivotal role in cell cycle alterations and apoptosis, common cellular events observed in the heat-induced teratogenesis model, might be upregulated with the induction of HSP90s.
Therefore, initial studies were conducted to better understand the patterns of p53, HSP90, and HSP90ß expression and their relationship in terms of coregulation after exposure to heat at levels that cause malformations (42°C for 15 min in vitro in whole embryo culture). Expression at different post-heat time points was compared with that of control embryos (37°C for 15 min) using immunohistochemistry. Our data indicate that HSP90 and p53, but not HSP90ß, are induced by heat in mammalian embryos. Moreover, HSP90 and p53 expression profiles are different in terms of their location in the embryo and intracellular localization. Colocalization experiments suggest that few cells express both p53 and HSP90 in heat-treated embryos. Therefore, HSP90 is not likely to play a regulatory role for p53 expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Sprague-Dawley derived CD rats (Charles River, Kingston, NY) were placed in polypropylene cages with stainless steel lids and were maintained in controlled conditions of light (12 h light, 12 h dark), temperature, and humidity. Food and water were provided ad libitum throughout the study. Nulliparous females were mated singly with a proven male on the afternoon of proestrus. The day of finding sperm was designated as GD 0.
Whole embryo culture.
GD 10 embryos were isolated and whole embryo culture was performed according to Kimmel et al.(1993b). Briefly, GD 10 embryos were isolated in Waymouth's medium (pH 6.9–7.0), and Reichert's membrane was opened to expose the visceral yolk sac. Embryonic heart beat, yolk sac circulation, flexion and position of the neural folds were noted as general signs of viability and developmental stage. Embryos used for culture were at or near the time of neural fold apposition (10–12 somites in our laboratory). Incubation vials (20 ml) containing 2 ml of heat-inactivated undiluted male rat serum were gassed with 15% O2/5% CO2/80% N2 for two 2-min periods, separated by 60 min. Embryos were equilibrated in the vials (1–2 embryos per vial) for 1–2 h in a 37–38°C incubator with rotation at 30–40 rpm (Roto-Torque Rotator, Cole Palmer, angled to maximize serum surface area) preceding heat exposure.
Exposure.
Vials containing embryos were exposed either to 37°C (control) or to 42°C in a water bath for 15 min and returned to a 37°C incubator/rotator. Embryos were removed from the incubator after 0.5, 1, 3, or 5 h and surrounding membranes were removed.
Immunohistochemistry.
Embryos were fixed in 10% neutral buffered formalin for 1 h and transferred to 70% EtOH. Following paraffin embedding, 5-micron sections were taken for immunohistochemical localization of p53, HSP90, and HSP90ß expression. Immunostaining for p53 was done using UniTect Immunohistochemistry detection systems according to the manufacturer's protocol (Oncogene Science, Uniondale, NY). After peroxidase blocking, deparaffinized slides were boiled (constant boiling in the microwave under low level of heat) in 10 mM citrate buffer (pH 6.0) for 10 min. Slides were blocked with normal rabbit serum for 1 h at room temperature or overnight at 4°C and were treated overnight with sheep polyclonal antibody raised against recombinant p53 (1:500 in 1% BSA made in PBS, Calbiochem, San Diego, CA). Normal sheep serum was used for negative controls. After washing, the slides were incubated with biotinylated rabbit anti-sheep IgG secondary antibody (1:10000 made in PBS) for 1 h. Slides were then washed, and incubated with ABC reagent (avidin:biotinylated horseradish peroxidase complex) for 30 min. p53 was visualized by the addition of DAB (diaminobenzidine tetra-hydrochloride, Dako, Carpinteria, CA) for 8 min. The sections were counterstained with hematoxylin for 10 s, dehydrated, and mounted.
Immunolocalization of HSP90 and HSP90ß was done using a LSAB2 immunodetection kit specific for rat specimens (Dako, Carpinteria, CA) according to the manufacturer's protocol. Slides were blocked with 3% H2O2 followed by washing and 30 min incubation at room temperature with rabbit polyclonal antibody raised against a synthetic peptide from the N-terminal region of mouse HSP90 (the area most different in amino acid sequence from HSP90ß; 10 mg/ml in PBS, Affinity Bioreagents, Golden, CO). After washing, slides were incubated with secondary antibody for 10 min. Slides were washed and incubated with streptavidin peroxidase for 10 min. Positive stain was visualized by the addition of DAB for 8 min. The sections were counterstained with hematoxylin for 10 s, dehydrated, and mounted. The relative intensity of immunostaining was graded blind to treatment on a 0–3 scale (0 = no stain or very little; 1 = low; 2 = medium; 3 = high) by two individuals using a light microscope (Olympus, Nikon Optiphot 2, Tokyo, Japan).
Dual-labeling for HSP90 and p53.
Embryos were fixed, processed, and sections made as described earlier. Slides were boiled in the microwave and blocked for peroxidase as described previously, then blocked with normal donkey serum (10 drops in 10 ml of PBS; Jackson Immunoresearch, West Grove, PA). Slides were incubated with sheep anti-p53 polyclonal antibody (Oncogene Science, Uniondale, NY) diluted 1:500 with 0.1% BSA in PBS (Jackson Immunoresearch, West Grove, PA) overnight at 4°C. After washing with buffer, slides were incubated for 1 h at room temperature with 1:2500 diluted biotin-SP-conjugated donkey antisheep antibodies (Jackson Immunoresearch, West Grove, PA). After washing, 1 mg/ml of streptavidin alexa 594 (Molecular Probes, Eugene, OR) was added for 30 min. After washing, the same slides were used for the detection of HSP90. Slides were again blocked with normal donkey serum followed by the addition of 1:100 dilution of rabbit anti-HSP90 polyclonal antibody (Affinity Bioreagents, Golden, CO) for 1 h at room temperature. FITC-conjugated donkey anti-rabbit antibody (1:100 dilution, Jackson Immunoresearch, West Grove, PA) was added for 1 h at room temperature after washing. Slides were then washed, rinsed in the equilibration buffer, a few drops of slowfade antifade reagent with 50% glycerol were added, and the slides were cover-slipped. Slides were analyzed under an Axioscope 20 fluorescence microscope (Zeiss, Germany) for red fluorescence (p53) and green fluorescence (HSP90). Images of the sections were overlaid using Adobe photoshop 5.5 (Adobe Systems, Inc) for the co-localization of p53 and HSP90 signals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Control Embryos Express Very Low Levels of p53
As shown in Table 1
, p53 expression was minimal in all of the tissues studied under control (37°C) incubation conditions (Figs. 1a and 1c). p53 expression was clearly nuclear and limited to a few cells. In the optic vesicle, the stain was limited to random epithelial cells (Fig. 1a). p53 staining was seen in more cells in the neuroepithelium, compared to optic vesicle. This may be due to the more compact nature of the neuroepithelial cells than other cell types in the embryo (Fig. 1c). In somites and presomitic mesoderm of control embryos, there was little or no expression as compared to that in the optic vesicle and neuroepithelium (Figs. 2a and 2c). There was no p53 expression in mature (rostral) somites, but a few cells in immature (caudal) somites expressed p53 in some of the control embryos (Table 1). The heart did not show any p53 expression in control or heat-treated embryos.
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Hyperthermia Induces p53 Expression
Following hyperthermic exposure to 42°C for 15 min, p53 expression in the optic vesicle increased dramatically at all postheat exposure times and was seen only in the nucleus (Fig. 1b
, 3 h postexposure). Induction of p53 was more evident in the neuroepithelium, especially in the rostral neuroepithelium, than in any other region of the embryo following hyperthermia (Fig. 1d, 3 h postexposure). In somites, increased p53 expression was seen but was not as extensive as in neural areas (Figs. 2b and 2d, 3 h postexposure). The optic vesicle, rostral neuroepithelium, and mature (rostral) somites all exhibited an increased p53 response within 0.5–1 h after the exposure. Maximum expression was observed at 3–5 h. Time periods beyond 5 h were not investigated. A rostral-caudal gradient in p53 expression in response to heat was apparent in the neural epithelium and somites. In the mid-neuroepithelium, moderate immunostaining was observed at 1 h postexposure, never reached the intensity of staining observed in rostral neuroepithelium, and decreased to low levels by 5 h. In the caudal neuroepithelium, there was only low staining throughout the 5 h postexposure period. Whether this minimal response was greater than in the controls was unclear, given the subjective nature of the grading system. The somites and presomitic mesoderm exhibited a similar rostral-caudal pattern with the expression being somewhat greater in the more mature (rostral) somites, less in the epithelial (immature caudal) somites, and only minimally expressed in the presomitic mesoderm. Within mature somites, the dermatomyotome appeared to show a slightly greater expression over 1–5 h post-exposure than the sclerotome.
HSP90 Expression Does Not Follow the Same Pattern as p53 Expression
Under control conditions, there was low-to-moderate expression of HSP90 in all tissues examined (Table 2). HSP90 expression was clearly cytoplasmic and not much different among the different regions examined in control embryos (Figs. 3a, 3c, 4a, and 4c). Heat exposure was associated with an increased expression of HSP90 throughout the embryo (Figs. 3b, 3d, 4b, and 4d, 3 h postexposure). Expression was mostly in the cytoplasm. However, expression was also observed in the nucleus in a few cells. Induction levels of HSP90 were clearly higher in optic vesicle and neuroepithelium compared to the somites (Figs. 3b and 3d). A slight increase in HSP90 expression after heat exposure was sometimes evident in the somitic area (Figs. 4b and 4d), but was not consistent (Table 2). There was a suggestion of minimal expression in the heart of both control and heat-exposed embryos (Table 2).
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The rostral-caudal gradient observed for p53 expression after heat exposure was not as evident for HSP90
expression. In the neuroepithelium, expression in the rostral and mid regions was similar, with time-dependent expression increasing to 3 h posttreatment and decreasing somewhat at the 5-h time point. Expression in the caudal neuroepithelium was lower than in either the rostral or mid-neuroepithelium, and it was unclear whether expression in the caudal region was different from that observed in controls. In the somites and presomitic mesoderm, there was no rostral-caudal gradient in HSP90 expression. Expression over that found in controls was minimal in all regions, but there was a consistent small increase at 3 h (somites) to 5 h (presomitic mesoderm) postexposure.
HSP90ß Expression Was Not Changed with Heat Exposure
Basal levels of HSP90ß expression were high and present in all areas of both control and heat-treated embryos (Table 3). The expression of HSP90ß was both cytoplasmic and nuclear. There was no difference in the HSP90ß expression profile in different regions of the embryo, compared to that seen with HSP90 expression.
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Colocalization of p53 and HSP90
Colocalization experiments were conducted to examine whether heat induces p53 and HSP90
by the same cells in the embryo. Few cells in the rostral neuroepithelium expressed both proteins (Fig. 5, 3 h postexposure). This can be clearly seen as the p53 expression is nuclear (red) and the HSP90 expression is cytoplasmic (green). However, HSP90 expression was ubiquitous while p53 expression showed a rostral to caudal gradient, and no correlation was observed between the two in the heat-exposed embryos 3 h postexposure, a time of maximal expression for both proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study clearly demonstrates that the tumor suppressor protein p53 is induced in GD 10 embryos exposed in vitro to 42°C for 15 min. Several recent studies suggest that p53 plays a crucial role in embryonic development; p53 overexpression induces growth arrest, DNA repair activity associated with the G0/G1 check point, or apoptosis occurring either through the G0/G1 check point or S phase (for review, see Almog and Rotter, 1997
). Null or overexpression of p53 can be teratogenic to the developing embryo. For example, p53 knockout mouse embryos developed abnormally and had neural tube defects such as exencephaly (Sah et al., 1995). Overexpression of p53 severely disrupted germ layer formation and neurulation in Xenopus (Hoever et al., 1994), and formation of the lens in mice (Nakamura et al., 1995). 2`-Deoxyadenosine, a DNA-damaging drug, induces p53 and causes apoptosis in developing lens in day 8 mouse embryos with associated eye defects in day 17 mouse fetuses (Wubah et al., 1996). Bolaris et al.(2001) reported induction of p53 and apoptosis in the developing rat brain after exposure to X-rays on GD 15 or 17. Therefore, p53 plays an important role in development, and induction of p53 by heat exposure may play a role in heat-induced teratogenesis in rats.
A number of teratogenic effects associated with hyperthermia have been observed in humans and animals. The defects seen depend heavily on the temperature and duration of exposure as well as on the timing of exposure; central nervous system (CNS) malformations have been the most commonly reported defects in all species (Graham et al., 1998). CNS defects occur after exposure of GD 9 rat embryos in vitro to relatively higher temperatures and longer periods of time than used in this study (43°C for 15 min; 42°C for 60 min; e.g., Mirkes, 1985). In our studies, GD 10 embryos exposed at 42°C for 15 min in vitro showed delays in several growth parameters including forebrain development as well as somite defects (Kimmel et al., 1993b). Breen et al.(1999), using a similar heat regimen and TUNEL staining, showed increased apoptosis and reduced mitotic figures in GD 10 rat embryos in vitro, particularly in the neuroepithelium and optic vesicle. Because of the increase in apoptosis seen in the neural tube after heat exposure, it might be tempting to conclude that p53 plays a role in heat-induced neural tube defects. However, damage to the CNS after exposure on GD 10 appears to be repaired, at least at the macroscopic level, because only a few cases of hydrocephalus and microphthalmia were seen in PND 3 pups exposed in vivo on GD 10 (Kimmel et al., 1993a). To the best of our knowledge, the study reported here is the first to show that heat induces p53 expression in the mammalian embryo and that there is a rostral-caudal gradient of p53 expression that is extensive in the optic vesicle and rostral neuroepithelium but somewhat less in the mid and caudal neuroepithelium. The implications of this pattern of p53 expression are not known.
Heat exposures to GD10 embryos (10–12 somite stage) in vivo or in vitro were shown to cause somite defects and skeletal malformations (Cuff et al., 1993; Kimmel et al., 1993a,b). Defects seen in thoracic vertebrae 3–5 (formed from somites 15–18) in offspring of heat-exposed GD 10 pregnant rats suggest that somites forming just after the time of heat exposure are more vulnerable than those already formed (Breen et al., 1999; Cuff et al., 1993). If the p53 induced apoptosis and/or cell cycle arrest mechanism is involved in heat-induced vertebral defects, we might expect to see more p53 expression in the presomitic mesoderm, which buds off into the immature epithelial somites. Interestingly, p53 expression in the presomitic mesoderm was less than in more mature (rostral) somites that had already formed and differentiated. This suggests the involvement of other mechanisms in heat-induced vertebral malformations.
There are ample data showing that heat induces heat shock proteins such as HSP70, HSP90, and HSP27 in embryos, and it has been suggested that these proteins may provide protection (Edwards et al., 1997). However, a dose-dependent increase in teratogenic effects and concomitant induction of heat shock protein expression does not suggest a protective role of HSPs against heat-induced teratogenic effects. Thus, the mechanism responsible for heat-induced teratogenesis and the role of heat shock proteins is not clear. Aligue et al.(1994) reported that HSP90 controls the cell cycle by regulating Wee1, which activates negative cell cycle kinases. Studies have demonstrated HSP 90 expression in embryogenesis and its alteration with heat or other environmental stress. For example, HSP90 mRNA levels were induced in a dose-dependent manner in GD 9.5 rat embryos exposed to 42°C for 10 min and 43°C for 7.5 min (Edwards et al., 1997). HSP90 was induced in the G0 phase of the cell cycle in GD 9.5 rat embryos treated for 42°C for 10 min and cultured for 1 h after heat (Walsh et al., 1994). No change in HSP90 was observed in GD 10–12 rat embryos when treated with caffeine in vivo (Wilkinson and Pollard, 1993). However, none of these studies differentiated the tissue expression and alteration for the HSP90 isoforms with heat exposure.
In this study, we have demonstrated that heat exposure at 42°C for 15 min induces HSP90 but not HSP90ß in GD 10 rat embryos in vitro. HSP90 expression in the present study does not follow the rostral-caudal pattern of expression seen for p53. HSP90ß is abundantly expressed throughout the embryo in both controls and heat-exposed embryos and is not affected by exposure. HSP90 induction by heat in the present study appears generally throughout the embryo, but is more highly induced in the optic vesicle, rostral, and mid brain. In contrast, HSP90 was expressed in the zebrafish and chick only in the myogenic cells of somites in controls, but was expressed throughout the embryo after heat exposure (Sass and Krone 1997). Therefore, the role of HSP90 may be different in normal rodents and in zebrafish, but may be similar after heat exposure.
As indicated earlier, HSP 90 and HSP70 have been known to associate with mutant and native forms of p53 and may be involved in the regulation of p53 conformation (Dasgupta and Momand, 1997; Ohnishi et al., 1995; Selkirk et al., 1994). Moreover, wild type p53 folding for proper function is temperature sensitive and can be affected at 41°C and above (Hainaut et al., 1995). Therefore, we compared the expression profiles of heat inducible HSP90 and p53 at different postheat time points. Kinetics and colocalization experiments indicate that p53 and HSP90 protein expression profiles are different, both in terms of the rostral-caudal gradient pattern seen for p53 but not HSP90 and because very few cells were observed showing both p53 and HSP90 proteins. Therefore, our data do not suggest a role of HSP90 in the regulation and stabilization of p53 in heat-treated embryos. Further studies are needed to establish the relationship between these proteins and their functional implications in heat-induced teratogenesis.
In summary, data from the present study show that heat exposure of GD 10 embryos to 42°C for 15 min induces p53 and HSP90 expression. However, the patterns of expression do not correlate with the high incidence of somite and skeletal defects that are seen after GD 10 heat exposure. Even though p53 expression and apoptosis were extensive in the neuroepithelium, embryos exposed on GD 10 appeared to recover from these effects because very few CNS and eye defects were observed in offspring exposed at this time. Due to the observation of high p53 expression in heat-treated embryos in a rostral-caudal gradient manner, it would be tempting to speculate that more differentiated cells are more sensitive to heat-induced DNA damage. However, the effect of heat on somites seems to be in the presomitic mesoderm or immature (caudal) somites. The role of HSP90 in the defects seen is also questionable, since expression was ubiquitous in the embryo. Future studies will explore further the role of these and other proteins in the developmental defects seen after heat exposure.
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ACKNOWLEDGMENTS |
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The work described in this paper was conducted under an interagency agreement between the U.S. Environmental Protection Agency and the U.S. Food and Drug Administration. C.A.K. and G.L.K. work as guest workers at the U.S. FDA on these studies. We acknowledge Dr. Mel Stratmeyer, Chief, Health Sciences Branch, LSD/OST/CDRH/FDA for his support and encouragement of this work.
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NOTES |
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1 Present address: Schering-Plough Research Institute, Reproductive and Specialized Toxicology, 144 Route 94, P.O. Box 32, Lafayette, NJ 07848–0032.
2 To whom correspondence should be addressed at U.S. EPA, NCEA-W (8623D), ORD, U.S. EPA, Ariel Rios Building, 1200 Pennsylvania Ave., NW, Washington, DC 20006. Fax: (202) 565-0078. E-mail: kimmel.carole{at}epa.gov.
The views expressed in this paper are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency or the U.S. Food and Drug Administration.
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