Teratogenesis, Carcinogenesis, and Mutagenesis 11:235-244 (1992)

Heat Shock Proteins in Human and Mouse Embryonic Cells After Exposure to Heat Shock or Teratogenic Agents Ken-ichi Honda, Takumi Hatayama, Ken-ichi Takahashi, and Munehiko Yukioka Departments of Biochemistry (K.-i. H., TH., M.Y.) and Department of Anatomy (K.-i. T), Osaka City University Medical School, Osaka, Japan In human chorionic villus tissue at the 10-17th week of a normal pregnancy, heat shock proteins (hsp70, hsp73, hsp85, and hspl05) were induced in vitro by a heat shock or by exposure to sodium arsenite or cadmium chloride. In dispersed cells of the whole mouse embiyo on the 1 lth day of development, heat shock proteins (hsp73 and hspl05) were induced by a heat shock or by exposure to sodium arsenite, but not by exposure to cadmium chloride. After a maternal hyperthermia or an intraperitoneal injection of sodium arsenite or cadmium chloride into a pregnant mouse, heat shock proteins accumulated in the embryo on the 9th day of development, especially in the neuroepithelial tissue. The :significanceof heat shock proteins in the embryo is discussed. o 1992 Wiley-Liss, Inc. Key words: hyperthermia, arsenic, cadmium, chorionic villus tissue, mouse embryo, neuroepithelial tissue

INTRODUCTION

Induction of heat shock proteins by heat or certain chemical agents occurs in a wide range of living organisms [ 11. Heat shock proteins are also induced by other environmental stresses like anoxia or trauma [2,3]. Some heat shock proteins are constitutively synthesized at high levels during certain stages of cell maturation and may have physiological functions in the cells [4]. Heat shock proteins with molecular weights in the range of 68,000-73,000 with a common antigenicity throughout evolution are referred to as hsp70s [5,6]. HeLa cells

Ken-ichi Honda, M.D., is now at Department of Obstetrics and Gynecology, Osaka City University Medical School, 1-4-54, Asashi-machi, Abeno-ku, Osaka 545, Japan. Address reprint requests and correspondence there. Takumi Hatayama is now at Department of Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607, Japan.

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have two kinds of hsp70s; one is strongly stress-inducible and has a smaller molecular weight as compared with the other, which is rather constitutively present in the cells [7]. In a previous study, we estimated the molecular weights of these hsp70s of HeLa cells as 70,000 and 73,000 [8], so we call them hsp70 and hsp73, respectively. In mammalian cells, a heat shock protein with a molecular weight of 105,000 (hspl05) is also inducible by heat or certain chemical agents [9]. The induction mechanism and the functions of heat shock proteins are not clearly understood. But it is likely that the intracellular accumulation of denatured proteins activates the synthesis of heat shock proteins in the cells [lo]. Hsp70 bound to ATP helps to solubilize the denatured proteins by ATP-derived hydrolysis [ 111. Furthermore, synthetic amount of heat shock proteins in the cells parallels with thermotolerance of the cells [I21 so that heat shock proteins are throught to have functions that protect cells from environmental stresses. In mouse embryos, the induction of synthesis of heat shock proteins by heat shock occurs from the blastocyst stage [13]. A mild heat shock which induces the synthesis of heat shock proteins protects rat embryos from teratogenic damage during a subsequent severe heat shock on the 9th day of development [ 14,151. Thus, heat shock proteins seem to protect embryos under thermal stress. In this paper, we show the induction of heat shock proteins by heat, sodium arsenite, or cadmium in human chorionic villus tissue and mouse embryo cells, We also studied the distributions of hsp70s and hspl05 in mouse embryos on the 9th day of development after a maternal hyperthermia or a maternal exposure to such teratogenic agents. MATERIALS AND METHODS Preparation of Human Chorionic Villus Tissues and Mouse Embryo Cells Human chorionic villus tissues were obtained from women undergoing therapeutic termination of normal pregnancy at 10- 17 weeks of gestation. The chorionic villus tissue was dissected into small pieces with scissors at 4°C in Eagle's minimum essential medium supplemented with 10%fetal calf serum. ICR mice (Japan SLC Inc.) were maintained on a 12 h light cycle and the day of positive vaginal smear was taken as the day 0 of development. The mouse embryos were removed from uterus on the 1 1th day of development and the cells of the whole embryo were dispersed by gentle pipetting at 4°C in the minimum essential medium supplemented with 10% fetal calf serum. Radioisotope Labeling of the Cells Under Various Conditions and Preparation of the Cell Extracts The cells of human chorionic villus tissue or the dispersed cells of whole mouse embryo were labeled with 20 p.Ci/ml [35S]methionine in methionine-deficient medium supplemented with 10% fetal calf serum under various conditions. For a heat shock at 42"C, the cells were incubated in the medium containing [35S]methionine for 3 h in a C02incubator at 42°C. For a heat shock at 45"C, the cells were incubated in the medium prewarmed at 45"C, kept at 45°C for 10 min by immersing the culture dish in a water bath prewarmed at 45"C, and then incubated with the fresh medium containing ["SImethionine for 3 h in a COz incubator at 37°C. For drug treatments, the cells were incubated in the medium containing [35S]methionine, in the presence of 50 p M

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sodium arsenite, or 80 p M or 400 p M cadmium chloride for 3 h in a C 0 2 incubator at 37°C. The labeled cells were washed twice with cold phosphate-buffered saline (PBS; 140 mM NaCl and 50 mM sodium phosphate, pH 7.4) and homogenized with a Teflonglass homogenizer in the hypotonic buffer (20 mM Tris-acetate, 20 mM NaCl, 0.1 mM dithiothreitol, 0.1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride, pH 7.5). The homogenate was centrifuged at 12,OOOg for 20 min at 4°C. To the supernatant, sodium dodecyl sulfate (SDS) was added to a final concentration of 0.1%, and the mixture was boiled for 2 min and stored at - 20°C. Two-DimensionalGel Electrophoresis

The cell extracts (0.5 mg of non-radioactive proteins or 200,000 cpm of [35S]methionine-labeled proteins) were treated with RNase A and the proteins were separated by two-dimensional gel electrophoresis [ 161. For the first dimension, an isoelectric focusing gel containing 1.6% pH 6-8 ampholytes and 0.4% pH 3.5-10 ampholytes was used. For the second dimension, a 10% polyacrylamide SDS slab gel was used. After electrophoresis, SDS slab gels were stained with Coomassie brilliant blue R and destained. For the [35S]methionine-labeledproteins, the gels were further processed with Amplify (Amershani Inc.) and fluorographed at - 80°C. Productionof Anti-hsp70s and Anti-hspl 05 Antisera

Anti-hsp70s antiserum was produced by immunizing rabbits with hsp70 and hsp73 of human chorionic villus cells. Human chorionic villus tissues obtained from a woman undergoing therapeutic termination of a normal pregnancy at 17 weeks of gestation were homogenized in the hypotonic buffer, and centrifuged at 105,OOOg for 1 h. The supernatant was chromatographed on a column (2 cm X 60 cm) of DEAE-Sepharose CL 6B (Pharmacia Fine Chemicals). Hsp70 and hsp73 were further separated by twodimensional gel electrophoresis. They were identified in comparison with radioisotopelabeled proteins on a two-dimensional gel. The Coomassie blue-stained spots of hsp70 and hsp73 were cut from the gels, neutralized, and homogenized with a Teflon-glass homogenizer. The homogenate was emulsified with Freund's complete adjuvant for the first immunization and with incomplete adjuvant for the second and third immunizations. Emulsified proteins were injected subcutaneously into rabbits three times with 3 week intervals between injections. The antiserum reacted to human hsp7Os and also to mouse hsp70s (data not shown). Anti-mouse hspl05 antibody was produced by immunizing rabbits with hspl05 of mouse FM 3A cells. The antiserum reacted to hspl05 and 42°C-specific heat shock protein of mouse cells [ 171. The anti-hsp70s or the anti-hspl05 serum was treated with ammonium sulfate at 50% and then 33% saturation and the precipitated IgG fraction was used for the immunohistochemical study. Treatment of Pregnant Mouse and lmmunohistochemicalStudy of the Embryos

On the 8th day of development, the pregnant mouse was stressed by a heat shock at 42"C, or by an intraperitoneal injection of sodium arsenite or cadmium chloride. For a heat shock at 42"C, the pregnant mouse was anesthesized with an intraperitoneal injection of pentobarbital at the dose of 30 mg/kg of body weight. The mouse was wrapped with a flexible vinyl bag except for the head, immersed intermittently in a water bath kept at 45°C to keep the rectal temperature at 42°C for 15 min, and then

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recovered at room temperature. For drug treatments, sodium arsenite in 1 ml of PBS or cadmium chloride in 0.2 ml of 0.85% NaCl was injected intrapentoneally into the pregnant mice, at the dose of 10 or 5 mg/kg of body weight, respectively. The decidual capsules including the embryo were removed from the pregnant mouse on the 9th day of development, 16 h after a maternal hyperthermia or a drug treatment, and were fixed in periodate-lysine-paraformaldehydesolution for 4 h at 4°C. After being washed successively with PBS containing 10, 15, and 20% sucrose, the decidual capsules incliiding the embryo were embedded in Tissue-Tek (Miles, Inc.) and stored at -80°C. The embedded tissues were sliced 8 p m thick and set on albumencoated glass slides, which were allowed to dry in the air and immersed in 98% methanol with 0.6% H202 for 30 min at room temperature to inactivate the cellular peroxidase. After incubation with 1% bovine serum albumin in PBS, the tissues were allowed to react with anti-hsp70s IgG or anti-hsp105 IgG, and then with anti-rabbit IgG antibody conjugated with peroxidase. The immune complexes were visualized in 50 mM Tris-HC1 buffer (pH 7.6) containing 0.05% diaminobenzidine and 0.01% H202.Preimmune rabbit IgG produced no immune complexes in the samely processed tissues. RESULTS

The synthesis of heat shock proteins in a human chorionic villus tissue at the 1 1th week of a normal pregnancy is shown in Figure 1. By two-dimensional gel analysis, the heat shock proteins of the chorionic villus cells were exactly the same as those of human HeLa cells [8]. Hsp70, hsp73, hsp85, and hspl05 were constitutively synthesized in an unstressed tissue and the synthesis of these heat shock proteins increased by the heat shock at 42" or 45"C, or by the exposure to 50 pM sodium arsenite, or 80 and 400 p M cadmium chloride. Four hundred micromolar cadmium chloride induced heat shock proteins more than 80 (IM cadmium chloride. The 42°C-specific heat shock protein which is synthesized only during the incubation of mammalian cells at 42°C [8] and have a common antigenicity with hspl05 [17] was also synthesized in a human chorionic villus tissue during the incubation at 42°C. The synthetic pattern of heat shock proteins in a human chorionic villus tissue was the same through the 10- 17th week of a normal pregnancy. Heat shock proteins synthesized in the dispersed cells of whole mouse embryo is shown in Figure 2. By two-dimensional gel analysis, the heat shock proteins of the mouse embryo cells were exactly the same as those of mouse FM 3A cells [ 171. We used the mouse embryos on the 1Ith day of development, because the mouse embryos on the 9th day of development were too small to get the cell extract. Hsp73 and hspl05 were constitutively synthesized in the unstressed cells, and the synthesis of these heat shock proteins increased by the heat shock at 42°C or by the exposure to 50 pM sodium arsenite, but not by the exposure to 400 p M cadmium chloride. The heat shock protein with the molecular weight of 85,000 (hsp85) that is constitutively abundant in the cells was not detected, probably because the excess amount of proteins were loaded on the isoelectric focusing gel. We further examined immunohistochemically the effects of maternal stresses on the distribution of these heat shock proteins in the mouse embryos at the critical stage of neural tube formation. Hsp70s were found throughout the embryo of untreated preg-

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Fig. I . Induction of heat shock proteins in human chorionic villus tissue. The cells of human chorionic villus tissue at the 11th week of a pregnancy were labeled in vitro with 20 FCi/ml [35S]methioninefor 3 h at 37" C (A), for 3 h at 42"C (B), for 3 hat 37°C after heat shock at 45°C for 10 min (C), for 3 h at 37°C in the presence of SO pM sodium arsenite (D), 80 FM cadmium chloride (E), or 400 (LMcadmium chloride (F). The labeled proteins were separated by two-dimensional gel electrophoresis and detected by fluorography. The two-dimensional gels are shown with the acidic end to the right. a, b, c, d, and e indicate hsp70, hsp73, hsp8.5, hsplOS, and 42"C-specific heat shock protein, respectively.

nant mouse on the 9th day of development, but not accumulated at high concentration in any particular place (Fig. 3). The distribution of hsp70s in the embryo was not altered by the pentobarbital anesthesia of a pregnant mouse (data not shown). After the exposure of a pregnant mouse to a heat shock at 42°C for 15 min, sodium arsenite, or cadmium chloride, hsp70s increased throughout the embryo and accumulated at high concentration in the neuroepithelial tissue of the embryo (Fig. 3). HsplO5 was also found throughout the embryo of the untreated pregnant mouse on the 9th day of development, but did not accumulate at high concentration in any particular place (Fig. 4). The distribution of hspl05 in the embryo was not altered by pentobarbital anesthesia of a pregnant mouse (data not shown). After the exposure of a pregnant mouse to a heat shock at 42°C or sodium arsenite, hspl05 increased through-

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Fig. 2. Induction of heat shock proteins in mouse embryo cells. Mouse embryo cells obtained from a pregnant mouse on the 1 1th day of implantation were labeled in vitro with 20 @/ml [35S]methioninefor 3 h at 37°C (A), for 3 h at 42°C (B), for 3 h at 37°C in the presence of 50 pM sodium arsenite (C), or 400 IJ.M cadmium chloride (D). The labeled proteins were separated by two-dimensional gel electrophoresis and detected by fluorography. b and d indicate hsp73 and hspl05, respectively.

out the embryo and accumulated at high concentration in the neuroepithelial tissue of the embryo. HsplO5 increased in the neuroepithelial tissue of embryos after the exposure of a pregnant mouse to cadmium chloride. DISCUSSION We first examined the inducibility of heat shock proteins in a human chorionic villus tissue through the 10- 17th week of a normal pregnancy. Hsp70, hsp73, hsp85,

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Fig. 3. Distribution of hsp70s in mouse embryos on the 9th day of development. Pregnant mice untreated (A), heat-shocked at 42°C for 15 min (B), or injected intraperitoneally with 10 mg/kg sodium arsenite (C) or with 5 mg/kg cadmium chloride (D) were killed 16 h after the treatment. The decidual capsules including the embryo were removed from the uterus, fixed, and reacted with anti-hsp70s IgC. The regions surrounded with arrowheads are the neuroepithelial tissues.

and hsp 105 were constitutively synthesized and the synthesis increased by heat shock, sodium arsenite or cadmium chloride. The induction pattern of these heat shock proteins in human chorionic villus tissue was the same through 10-17 weeks of gestation and also almost the same as that in HeLa cells of which origin is the human cervical cancer cells. In mouse embryo cells on the 1lth day of development, hsp73 and hspl05 were constitutively synthesized and the synthesis increased by heat shock or sodium arsenite. Cadmium chloride did not induce heat shock proteins in dispersed mouse embryo cells. As the absorption of cadmium chloride into the cells depends on cell species and environmental conditions [ 181, cadmium chloride may be hardly absorbed into the dispersed mouse embryo cells. After we confirmed the induction of heat shock proteins in mouse embryos at the 11th day of development, we then studied the distribution of heat shock proteins immunohistochemically in mouse embryos at the 9th day of development. After hyperthemia (42°C for 15 min) of a pregnant mouse, hsp 70s and hsp 105 accumulated throughout the embryo, especially at high concentration in the neuroepithelial tissue. The fact that hsp 70s-mRNA is induced in neuroepithelial tissue after a heat shock of cultured rat embryos [ 191 supports our results. Severe hyperthermia of a pregnant mouse at the 8th or 9th day of development (43°C for 10 min, two times with

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Fig. 4. Distribution of hspl05 in mouse embryos on the 9th day of development. Pregnant mice untreated (A), heat-shocked at 42°C for 15 min (B), or injected intraperitoneally with 10 mg/kg sodium arsenite (C) or with 5 mg/kg cadmium chloride (D) were killed 16 h after the treatment. The decidual capsules including the embryo were removed from the uterus, fixed, and reacted with anti-hspl05 IgG. The regions surrounded with arrowheads are the neuroepithelial tissues.

a 6 h interval) results in a high incidence of embryo anomalies, of which exencephaly is the most common [20]. So, heat shock proteins seem to be induced more in heatsensitive organs. Intraperitoneal injection of sodium arsenite (10 mg/kg) [2 I] or cadmium chloride (5 mg/kg) [22] into a pregnant mouse at the 8th or 9th day of development also results in a high incidence of embryo anomalies, of which exencephaly is the most common one. Sodium arsenite inhibits thiol-dependent enzyme systems which are important for many cellular functions [23]. When cadmium is injected into a pregnant rat, the accumulation of cadmium in the embryo tissues is low, but zinc is deleted in the embryo tissues [24]. The cellular damage caused directly or indirectly by a teratogen are thought to result in malformation of the embryo. Heat shock proteins which are accumulated more in teratogen-sensitive neuroepithelial tissue may have some relation to the teratogenesis. In cultured rat embryos, a mild heat shock (42°C for 10 min) which is not teratogenic protects the embryos from teratogenic damage during a subsequent severe heat shock (43°C for 7.5 min) [14], so heat shock proteins seem to have protective roles for the embryo. Induction of heat shock proteins by either heat shock, sodium arsenite, or cadmium chloride correlates with the acquisition of thermotolerance of the cultured cells [25], suggesting protective functions of heat shock proteins in the cells. In Dro-

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sophila embryo cells, synthesis of heat shock proteins induced by a mild heat shock prohibits the disturbance of RNA splicing during a subsequent severe heat shock 1261. Thus, heat shock proteins induced in the embryo cells may be functioning to protect the development of the embryo. Furthermore, the induction of heat shock proteins in the chorionic villus cells may help us to evaluate the environmental risk for the embryo. ACKNOWLEDGMENTS

We thank Professor Tadashi Sugawa, Dr. Yuji Fujino, Dr. Akio Miyazaki, and other physicians of the Department of Obstetrics and Gynecology, Osaka City University Medical School, for their helpful advice. REFERENCES 1. Schlesinger MJ, Aliperti G , Kelley PM: The response of cells to heat shock. Trends Biochem Sci 7:222-2;!5, 1982. 2. Velazquez JM, Lindquist S: hsp 70: Nuclear concentration during environmental stress and cytoplasmic storage during recovery. Cell 36:655-662, 1984. 3. Fujio N, Hatayama T, Kinoshita H, Yukioka M: Induction of mRNAs for heat shock proteins in livers of rats afler ischemia and partial hepatectomy. Mol Cell Biochem 77: 173-177, 1987. 4. Banerji SS, Laing K, Morimoto RI: Erythroid lineage-specific expression and inducibility of the major heat shock protein HSP70 during avian embryogenesis. Genes Dev 1:946-953, 1987. 5 . Kelley PM, Schlesinger MJ: Antbodies of two major chicken heat shock proteins cross-react with similar proteins in widely divergent species. Mol Cell Biol2:267-274, 1982. 6 . Lindquist S: The heat-shock response. Annu Rev Biochem 55:1151-1191, 1986. 7. Welch WJ, Feramisco JR: Purification of the major mammalian heat shock proteins. J Biol Chem 257:1494.9-14959, 1982. 8. Hatayam,aT, Honda K, Yukioka M: HeLa cells synthesize a specific heat shock protein upon exposure to heat shock at 42°C but not at 45°C. Biochem Biophys Res Commun 137:957-963, 1986. 9. Subjeck .IR, Shyy TT: Stress protein systems of mammalian cells. Am J Physiol250:Cl-C17, 1986. 10. Ananthan J, Goldberg AL, Voellmy R: Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232522-524, 1986. 11. Lewis MJ, Pelham HRB: Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein. EMBO J 4:3137-3143, 1985. 12. Landry J, Bernier D, Chrktien P, Nicole LM, Tanguay RM, Marceau N: Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res 42:2457-2461, 1982. 13. Wittig S , Hensse S, Keitel C, Elsner C , Wittig B: Heat shock gene expression is regulated during teratocarcinoma cell differentiation and early embryonic development. Dev Biol96:507-5 14, 1983, 14. Walsh DA, Klein NW, Hightower LE, Edwards MJ: Heat shock and thermotolerance during early rat embryo development. Teratology 36:181-191, 1987. 15. Mirkes F’E: Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos. Dev Biol 119:llS-122, 1987. 16. O’Farrell PH: High resolution two-dimensionalelectrophoresis of proteins. J Biol Chem 250:4007-4021, 1975. 17. Honda K , Hatayama T, Yukioka M: Common antigenicity of mouse 42°C-specific heat-shock protein with mouse hsp 105. Biochem Biophys Res Commun 160:60-66, 1989. 18. Klug S, Planas-Bohne F, Taylor DM: Factors influencing the uptake of cadmium into cells in vifro. Hum Toxicol7:545-549, 1988. 19. Walsh DA, Li K, Speirs J, Crowther CE, Edwards MJ: Regulation of the inducible heat shock 71 genes in early neural development of cultured rat embryos. Teratology 40:321-334, 1989. 20. Webster WS, Edward MJ: Hyperthermia and the induction of neural tube defects in mice. Teratology 291417425, 1984. 21. Hood RD: Characteristics of arsenite as a teratogen. J Ala Acad Sci 42: 138-139, 1971.

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22. lshizu S , Minami M, Suzuki A, Yamada M: An experimental study on teratogenic effect of cadmium. Tokyo Women’s Med College J 42:744-752, 1972. 23. Ltonard A. Lauwerys RR: Carcinogenicity, teratogenicity and mutagenicity of arsenic. Mutdt Res 75:49-62, 1980. 24. Roelfiema WH, Roelofsen AM, Herber RFM, Copius Peereboom-Stegemdn JHJ: Cadmium and zinc concentrations in fetal and maternal rat tissues after parenteral administration of cadmium during pregnancy. Arch Toxicol62:285-290, 1988. 25. Li GC, Shrieve DC, Werb 2: Correlations between synthesis of heat-shock proteins and development of tolerance to heat and to adriamycin in Chinese hamster fibroblasts: heat shock and other inducers. In Schlesinger MJ, Ashburner M, Tissieres A: “Heat Shock From Bacteria to Man.” New York: Cold Spring Harbor Laboratory, 1982, pp 395-404. 26. Yost, HJ, Lindquist S: RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell 45:185-193, 1986.

Heat shock proteins in human and mouse embryonic cells after exposure to heat shock or teratogenic agents.

In human chorionic villus tissue at the 10-17th week of a normal pregnancy, heat shock proteins (hsp70, hsp73, hsp85, and hsp105) were induced in vitr...
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