TERATOLOGY 46:301-309 (1992)

Accumulation of Heat Shock Protein 72 (hsp 72) in Postimplantation Rat Embryos After Exposure to Various Periods of Hyperthermia (40°-43"C)In Vitro: Evidence That Heat Shock Protein 72 Is a Biomarker of Heat-Induced Embryotoxicity PHILIP E. MIRKES AND BARBARA DOGGETT Central Laboratory for Human Embryology, Division of Embryology, Teratology, and Congenital Defects, Department of Pediatrics, University of Washington, Seattle, Washington 98195

ABSTRACT A monoclonal antibody to the 72 kDa heat shock protein and Western blot analysis were used to determine the induction, accumulation and turnover of hsp 72 after day 10 rat embryos were exposed to elevated temperatures (40"-43"C) for various lengths of time (2.5 minutes to 18 hours). Embryos exposed to temperatures that exceed the normal culture temperature (37°C) by 4°C or more for as little as 2.5 minutes (43°C) or 15 minutes (41, 42°C) synthesized and accumulated detectable amounts of heat-inducible hsp 72. Hsp 72 could not be detected by Western blot analysis of proteins from embryos cultured at 40°C or below. Once induced, hsp 72 can be detected in embryos for 24-48 hours after they are removed from the hyperthermic conditions and returned to normothermic conditions. Our results also indicate that hsp 72 is induced by all hyperthermic exposures that induce alterations in rat embryo growth and development; therefore, hsp 72 is a potential biomarker for heat-induced embryotoxicity. o 1992 Wiley-Liss, Inc. Research published over the past 25 years has conclusively demonstrated that hyperthermia is teratogenic in all vertebrates studied to date (Edwards, '86; Warkany, '86) if exposure occurs a t the appropriate stage of development (Edwards, '88; Webster et al., '85) and if sufficient hyperthermic exposure occurs (Germain et al., '85). Although hyperthermia induces a variety of malformations in animals, the central nervous system is the most sensitive target for hyperthermia-induced alterations in development, and the most common malformations observed are exencephaly, microcephaly, and encephalocoeles (Edwards, '86). In addition to the teratogenic effects of hyperthermia, it is now well documented that exposure to elevated temperatures also induces a heat shock or stress response in organisms ranging from bacteria to man (see Nover, '91, for an extensive review). The heat shock response is characterized by the induction of a set of heat shock genes and the subsequent synthesis and accumu0 1992 WILEY-LISS, INC.

lation of a set of heat shock proteins, or hsps. One of the most prominent hsps, induced by heat and a variety of chemical stressors, is hsp 70. In mammalian cells hsp 70 is actually a family of at least 8 homologous proteins, some of which are constitutively produced in the absence of stress and others whose synthesis is induced by various stresses (Nover, '91). In this paper we use hsp 72 to denote the heat shock protein that is induced to relatively high levels by hyperthermia and is recognized by a commercially available antibody designated anti-hsp 72. Exposure to temperatures from 41°C to 43°C produces abnormal development in rat embryos exposed in vitro (Mirkes, '85). Moreover, as the exposure temperature is increased, the length of exposure required

Received December 18, 1991; accepted March 17, 1992. P.E. Mirkes is an Affiliate of Child Development and Mental Retardation Center, University of Washington, Seattle, WA.

302

P.E MIRKES AND B. DOGGETT

to disrupt normal development is decreased. More recent work has shown that acute exposures of hyperthermia (43°C) produce a rapid accumulation of hsp 70 mRNA (Walsh et al., '89; Harris et al., '91; Mirkes et al., '91) and the synthesis of a set of hsps, most prominent of which is hsp 70 (Mirkes, '87; Walsh et al., '87). Thus, hyperthermia-induced abnormal development of rat embryos exposed in vitro is associated with an hyperthermia-induced stress response. Given this background, we initiated studies using a monoclonal antibody to hsp 72 and Western blot analysis to determine 1) whether the accumulation of hsp 72 is correlated with abnormal development induced by exposure to temperatures of 40"C, 41"C, 42"C, and 43°C for various lengths of time and 2) the kinetics of hsp 72 accumulation and decay after various embryotoxic exposures of hyperthermia. Our results indicate that hsp 72 is a biomarker for hyperthermia-induced abnormal development and that hsp 72, once induced by an hyperthermia exposure, rapidly accumulates and remains in the embryo for 24-48 hours.

achieved by culturing conceptuses in an incubator set at 40°C. To assess the effects of exposure to various hyperthermic conditions on growth and development, embryos were exposed to a particular temperature for a specified amount of time, returned to a 37°C incubator, and cultured for a total of 24 hours. Embryos were then removed from culture and assessed for viability, growth, and development as described previously (Mirkes et al., '81). Protein determinations were made using the method of Lowry et al. ('51). Growth parameters were analyzed by one-way ANOVA with Dunnett's multiple comparisons (Zar, '84). To assess the effects of various hyperthermic exposures on the accumulation of hsp 72, the following exposure protocol was used. Conceptuses were exposed to temperatures of 41"C, 42"C, or 43°C for times ranging from 2.5 to 240 minutes. After these exposures, culture medium was rapidly returned to 37'C by swirling the culture bottle in ice. Culture bottles were then returned to the 37°C incubator and conceptuses were cultured for a total time of 5 METHODS hours, i.e., the time at elevated temperature Animals plus the time at 37°C equals 5 hours. We Primagravida Sprague-Dawley rats (Wis- chose to assess hsp 72 levels at this 5-hour tar-derived) were obtained from Tyler Lab- time point because preliminary analysis inoratories (Bellevue, WA) on day 5 or 6 of dicated that accumulation of hsp 72 was gestation. The morning following copula- maximal 5 hours after a 43"C, 15 minutes tion was designated day 0 of gestation. exposure. At this 5-hour time point, concepPregnant rats were maintained in the ani- tuses were removed from culture and emmal facility of the Central Laboratory for bryos were dissected from associated memHuman Embryology on a 14 hour lightilo branes and stored at -20°C. For the 40°C hour dark cycle and at a temperature of treatment, conceptuses were removed after 21°C. Access to food and water was ad libi- 1 hour from the 40°C incubator and then tum. cultured for 4 hours at 37°C before embryos were harvested for Western blot analysis, Embryo culture and stress induction Embryos exposed to 40°C for 5 or 18 hours On the morning of day 10 of gestation, were harvested for Western blot analysis conceptuses were removed from the uterus, immediately after the 5 or 18 hour expoexplanted and cultured in vitro as described sure. previously (New, '78; Mirkes et al., '84). Sample preparation and Conceptuses were cultured for 1 hour at one -dimensional PAGE 37°C and then exposed for various lengths of time to temperatures of 41"c, 42"C, or 43°C Embryos (10-12) were sonicated in susby placing the culture bottles in a water pension buffer (100 mM NaCl, 10 mM Trisbath equilibrated to the appropriate tem- HCl [pH 7.81, 1 mM EDTA) containing perature. Culture medium reached the ap- Aprotinin (1pgiml), lupeptin (1 pg/ml), and propriate temperature within 3-4 minutes. phenylmethylsulfonyl fluoride (PMSF) (100 The time a t which the culture medium pg/ml). After sonication, an equal volume of reached the desired temperature was con- gel loading buffer (125 mM Tris-HC1 [pH sidered zero time for determining the length 6.81, 4% SDS, and 10% glycerol) was added of exposure. Exposures a t 40°C were and the combined sample was boiled for 10

Hsp 72 ACCUMULATION

minutes. The sample was then cooled, centrifuged for 10 minutes a t 10,OOOg and the supernatant removed. Aliquots of this supernatant were taken and protein concentration determined using the Lowry assay (Lowry et al., '51). Bromophenol blue and beta-mercaptoethanol were added to the 10,OOOg supernatant to concentrations of 0.01% and 0.02%, respectively. Aliquots (10-20 pl) of this supernatant, containing 12.5 or 25 p,g of protein, were then subjected to slab gel electrophoresis (Laemmli, '70) andlor stored frozen a t -20°C. Western blot analysis Proteins resolved in slab gels from onedimensional gel electrophoresis were then transferred to Immobilon PVDF membranes (Millipore) using a semi-dry transfer apparatus. Membranes with transferred proteins were blocked with 2% milk in phosphate-buffered saline (PBS); washed with PBS, 0.1% Tween-20 for 1 hour; and then exposed to primary antibody (anti-Hsp 72, Amersham or anti-actin, Oncogene Science) overnight a t a dilution of 1:2,500 or 1:4,500, respectively. Membranes were then washed in PBS, 0.1% Tween-20 (three 15-minute washes) followed by incubation for 2 hours with secondary antibody (biotinylated sheep anti-mouse Ig, Amersham) at a dilution of 1:1,000. The membranes were then washed in PBS, 0.1% Tween-20 (three 15-minute washes) followed by incubation for 2 hours with streptavidin biotinylated peroxidase complex (Amersham) at a dilution of 1: 1,000. After three 15-minute washes in PBS, excess buffer was removed from the membrane by placing the membrane between sheets of chromatography paper. Antigen was visualized by covering the membrane with a piece of Enzygraphic Web (IBI) according to manufacturer's protocol. RESULTS

The effects of various hyperthermic exposures (temperature/time) on growth and development of rat embryos are presented in Table 1. Exposures a t 40°C for up to 18 hours had no effect on viability and did not induce abnormal development. Interestingly, exposures a t 40°C for 5 and 18 hours actually produced significantly increased growth compared with embryos maintained at 37°C as measured by increased crownrump length and embryo protein content. Exposures a t 41°C for 15 to 240 minutes

303

produced the following effects. Viability was only affected by exposure at 41°C for 240 minutes, while abnormal development was first observed after exposure at 41°C for 60 minutes. The induction of abnormal development is correlated with deficits in growth (crown-rump length; head measurement, i.e., A/B ratio; and embryo protein content) at exposures of 60 minutes or longer. Exposures at 42°C required a 30-minute exposure to elicit abnormal development and deficits in growth parameters. Finally, exposures a t 43°C produced abnormal development and reductions in growth after exposures of 2.5 minutes or longer. Thus, the thresholds for the induction of abnormal development and associated growth deficiencies are between 0 and 2.5 minutes a t 43"C, 15 and 30 minutes at 42"C, and 45 and 60 minutes a t 41°C. The morphology of embryos exposed above these threshold doses of hyperthermia is shown in Figure 1. For all three exposures-i.e., 43OU2.5 minutes (A), 42"C/30 minutes (B), and 4l0C/6Ominutes ((3)-embryo morphology is remarkably similar and obviously different from the morphology of a control embryo cultured a t 37°C (El. Embryos exposed at 40°C for 18 hours (D) are similar to control embryos and do not exhibit the abnormal head morphology characteristic of embryos exposed to higher temperatures. Embryos exposed to temperatures of 41°C or above are characterized by hypoplastic prosencephalon, edematous rhombencephalon, microophthalmia, and abnormal somite segmentation. With the exception of the somite abnormalities, the most obvious defects are limited to the head of the embryo. Using the exposures listed in Table 1, we next determined the presence and relative levels of hsp 72 by Western blot analysis. Figure 2A-D shows that the anti-hsp 72 monoclonal antibody used recognizes a distinct protein having a molecular weight of approximately 72 kD that is observed only in extracts from embryos exposed for various lengths of time to temperatures of 41"C, 42"C, or 43°C. Thus, this monoclonal antibody recognizes the heat-inducible member of the hsp 70 family. The level of heat-inducible hsp 72, based upon intensity of immuno-stained bands, increases with length of exposure to temperatures of 41"C, 42"C, and 43°C. In addition, the appearance of detectable levels of this heat-inducible hsp 72

(min)

Control 60 300 18 hr

iC")

37 40 40 40 37 41 41 41 41 41 37 42 42 42 37 43 43 43

No. of Embryos 31 30 30 30 32 32 33 32 31 22 23 25 28 19 37 31 37 38

of

100

0 13 59

-

0 90

0 0 0 0 0 0 0 40 82 100 0

abnormal3

viable

100 100 100 100 91 94 97 94 90 68 100 88 75 0 100 93 73 3.5

%

970

-

2.85 i .32 2.65 i .29* 2.60 i .29**

-

-

21.9 t 1.3 21.2 t 0.9* 19.1 i 1.2**

-

3.03 i .22 3.17 ? .36 3.15 ? .23 3.23 f .27** 2.91 t .25 2.94 ? .31 3.03 i .23 2.82 2 .31 2.77 2 .33 2.52 i- .32** 2.81 t .23 2.82 i .40 2.57 i .25*

(mm)

rump'

No. of somites' 23.4 i 1.8 23.8 i 2.1 23.8 t 1.6 24.0 ? 1.5 22.4 ? 1.6 22.7 2 1.1 21.5 2 1.2 20.4 i 1.7* 19.4 2 2.4* ND4 22.3 2 7.2 21.6 i 1.0 18.8 i 1.5**

Crown-

*112 * 38 104 f 26 99 * 26** -

111 f 17 110 2 36 127 i 22' 125 ? 29** 105 i 17 103 2 17 99 t 18 9 2 ? 16 91 i 15 79 t 14** 110 ? 2 1 104 f 15 90 12**

(pg/embryo)

Protein'

-

3.10 i .39 3.01 ? .35 2.80 ? .39

-

3.38 2 .32 3.41 ? .38 3.50 2 .30 3.56 ? .30 2.50 t 57 2.97 ? .46 3.16 i .22 2.85 2 .28 2.80 2 .29 ND 3.13 f .42 3.38 t .25 2.54 ? 2.2

imm)

A'

-

1.24 -+ .17 1.17 f .16 1.04 ? .17

-

1.40 t .13 1.41 ? .17 1.52 i .18 1.49 ? .13 1.00 ? .15 1.18 t .22 1.23 2 .16 1.05 2 .20 0.96 .08 ND 1.27 2 1.8 1.36 2 .12 0.90 ? .14

imm)

B'

various temperatures and lengths of exposure on growth and development of day 10 rat embryos

-

2.50 t .13 2.58 t .19* 2.68 t .18**

-

AIB',2 2.42 i . l l 2.42 i .06 2.30 k .11** 2.38 ? . l l 2.50 i .18 2.52 f .25 2.56 ? .18 2.69 2 .24** 2.90 2 .22** ND 2.46 ? .15 2.48 t .10 2.82 ? .30'*

*

"Denotes values significantly different from control, P