TERATOLOGY 46:191-200 (1992)

Genetic Differences in Heat-Induced Tolerance to Cadmium in Cultured Mouse Embryos Are Not Correlated With Changes in a 68-kD Heat Shock Protein CAROLYN M. KAPRON-BRk3 AND BARBARA F. HALES Department of Pharmacology and Therapeutics and Centre for the Study of Reproduction, McGill Uniuersity, Montreal, Quebec, Canada H3G 1 Y6

ABSTRACT Heat-induced cross-tolerance to cadmium was investigated in two inbred strains of mice, BALBic and SWV, using a whole embryo culture system. Embryos were exposed to a pretreatment of 5 min a t 43°C and subsequently to an embryotoxic concentration of cadmium, 1.75 pM. The two types of embryos responded differently to the heat pretreatment, as cross-tolerance was induced in SWV but not in BALBlc mice. In SWV embryos, prior exposure to 43°C for 5 min essentially eliminated the negative effects of cadmium on embryonic development and growth. However, in BALBic embryos, no protection was observed. The variation in development of cross-tolerance in embryos from the two strains of mice was not correlated with differences in the induction of a 68-kD heat-shock protein (hsp68). There was a rapid increase in this protein in both strains after the initial heat exposure but no excess induction in the SWV strain that developed tolerance. The induction of hsp68 is therefore not sufficient to elicit cross-tolerance, and other mechanisms are likely to be important in the protective response of the embryo. o 1992 Wiley-Liss, Inc.

It is well established that when most organisms are exposed to elevated temperatures they undergo a heat shock or stress response (Schlesinger et al., ,821, which includes changes in gene expression and the development of thermotolerance (Henle, '87). Postimplantation mouse and rat embryos growing in vitro react in a similar manner. A mild heat stress induces thermotolerance, characterized by resistance to the teratogenic, embryolethal, and growth inhibitory effects of a second, more severe exposure (Mirkes, '87; Walsh et al., '87; Kapron-Bras and Hales, '91). In recent studies it has been found that such protection extends to more than just a subsequent heat exposure. CD-1 mouse embryos that are thermotolerant have also been shown to be cross-tolerant to the embryotoxic agent, cadmium (Kapron-BrBs and Hales, ,911. The mechanisms of the thermotolerance and cross-tolerance responses in embryos are not known. In other systems, particularly cultured cells, there is extensive evidence that heat shock proteins (hsp's) may be playing important roles in the tolerance response (Hendrey and Kola, '91; Landry O 1992 WILEY-LISS. INC.

et al., '89; Li et al., '91). The induction and subsequent disappearance of hsp's when cells are exposed to heat and other stressful conditions correlate well with observations of tolerance (Li and Werb, '82; Gerweck and Epstein, '86;Landry et al., '82; Li, '83).However, there are also reports of experiments in which thermotolerance develops in the absence of stress protein induction (Fisher et al., '86; Boon-Niermeijer et al., '88) or a lack of thermotolerance is observed even when the stress proteins are induced (Landry and Chrktien, '83). In embryonic tissue, hsp's are also typically induced by a heat stress (Mirkes, '87; Walsh et al., '871, while at the same time there is a depression in overall protein synthesis (Bennett et al., '90). As in other systems, the hsp's can be grouped into families

Received October 2, 1991: accepted March 17, 1992. Address reprint requests to Dr. Barbara F. Hales, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond, Montreal, Quebec, Canada H3G 1Y6. Dr. Kapmn-BrBs is now at the Department of Cancer Fiesearch, Sunnybrook Health Sciences Centre, S-218, North York, Ontario, Canada M4N 3M5.

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of hsp27, hsp70, hsp90 and hspllO based on their apparent molecular masses (Subjeck and Shyy, '86). Although it has been suggested that these proteins may be involved in embryonic thermotolerance, this idea does not receive complete support; thermotolerance was observed not t o have been induced in rat embryos in a situation where hsp70 was found to be increased but glutathione was depleted (Harris et al., '91). Thus, further studies are needed to determine the mechanisms of tolerance in mammalian embryos. Genetically based differences in the response of embryos to harmful agents have proved useful in elucidating mechanisms of toxicity. Strain differences in the in vivo teratogenic response of mouse embryos to both heat and cadmium have been reported (Webster and Edwards, '84; Finnell et al., '86; Layton and Layton, '79; Kuczuk and Scott, '841, and it was therefore proposed that similar differences may exist with respect to the development of tolerance in the embryo. The present study examines the development of tolerance in two strains of mice, BALBic and SWV. These strains can be classified by their response to cadmium. BALB/c mice are relatively sensitive to cadmium-induced embryotoxicity (Layton and Layton, '79) and are also homozygous for a gene, cdm, which confers resistance to cadmium-induced testicular damage (Taylor et al., '73). SWV mice, on the other hand, are relatively resistant to cadmium-induced embryotoxicity (Kuczuk and Scott, '84) but to the best of our knowledge do not carry the cdm allele (Green, '89). The relationship between the cdm gene and embryotoxicity is not yet clear. The purpose of this study was to determine whether there are differences between BALB/c and SWV embryos in heat-induced tolerance to cadmium. The second objective was to examine the role of hsp's in the embryonic tolerance response by determining whether the induction of hsp68 (the mouse heat-inducible member of the hsp70 family) is correlated with the cross-tolerance response in the two strains. MATERIALS AND METHODS

Mice Male and female BALB/c mice (20 g) were purchased from Charles River, Canada (St. Constant, Quebec, Canada). They were used in experiments after a one-week acclimati-

zation period. Several breeding pairs of SWV mice were obtained from Dr. Alan Peterson, Ludwig Institute for Cancer Research, Montreal, and were subsequently maintained by brother-sister matings. Mice between 6 and 20 weeks of age were used for experiments. All mice were housed 1 to 5 per plastic cage with woodchip bedding and were given free access to water and Purina Mouse Chow. A constant temperature of 22?1"C was maintained, along with a cycle of 12 hr light, 12 hr dark. The mice were mated by placing 1 to 4 females with each male overnight. Those with vaginal plugs the next morning were considered to be in day 0 of gestation, and were grouped separately according to strain. Embryo culture Mouse embryos were cultured using standard techniques as previously described (Kapron-Bras and Hales, '91). In brief, pregnant females were killed by cervical dislocation at 11 A.M. on day 8 of gestation and uteri were removed to sterile Hank's Balanced Salt Solution (HBSS)(Gibco, Burlington, Ontario, Canada). Under aseptic conditions embryos were dissected free of decidual tissue and Reichert's membrane, with the visceral yolk sac and ectoplacental cone remaining intact. Embryos with 4 to 6 somites were cultured 3 per 60-ml bottle in 5.4 ml culture medium (90% immediately centrifuged, heat inactivated rat serum, 10% Tyrode's saline containing 10 U/ml penicillin G and 10 pgiml streptomycin (Gibco)). The bottles were gassed with a mixture of 5% 02,5% C 0 2 , 90% N2 at the start of culture and 20% 02,5% COe, 75% N2 a t 19 and 27 hr of culture. All groups of embryos were cultured in a 37°C incubator at 30 rpm for a total of 44 hr, except for the heat pretreatment period. At the end of culture the number of surviving embryos (with both heartbeat and blood circulation in the yolk sac) was determined, along with the number of malformed embryos among those that lived. Surviving embryos were scored according the method of Brown and Fabro ('81)) the number of somites was counted and the yolk sac diameter, crown-rump length and head length were measured. Testing for cross-tolerance The bottles containing embryos were allowed to equilibrate for 2 hr in the 37°C

GENETIC DIFFERENCES IN CROSSTOLERANCE

incubator. At the end of this period, one-half of the bottles that made up the pretreated group were heated in a 43.0+-0.2"C waterbath for 5 min. This treatment has been shown to be nontoxic but able to induce cross-tolerance to cadmium in CD-1 embryos (Kapron-Bras and Hales, '91). The other half of the bottles remained at 37°C. After this initial period of heating, all battles were maintained a t 37°C for 30 min. Then, one-half the pretreated group and one-half of the nonheated group were exposed to 1.75 pM cadmium for the rest of the culture period (41.5 hr). Previous experiments have shown that this concentration of cadmium, added as cadmium chloride in 0.1 ml of sterile distilled water, is embryotoxic to CD-1 mouse embryos in vitro (KapronBras and Hales, '91). Distilled water alone was added to the other half of each group. Thus, four treatment groups were obtained: a control group that remained a t 37°C for the entire culture period and was not exposed to cadmium; a heated group that was exposed to 43°C for 5 min; a cadmiumtreated group that was exposed t o 1.75 pM cadmium; and a heatlcadmium group that was first exposed t o 43°C for 5 min and subsequently to 1.75 pM cadmium.

Induction of hsp68 as indicated by Western blotting The induction of immunoreactive hsp68 in the two strains was examined by Western blotting (Towbin et al., '79; Burnette, '81) and identification with a monoclonal anti72kD heat-shock protein antibody that recognizes only the inducible form of hsp7O (human hsp72) and cross-reacts with rodent hsp7O (murine hsp68) (code RPN.1197, Amersham, Oakville, Ontario, Canada). The embryos were cultured 3 or 4 per 2 ml of culture medium and were again treated with heat or cadmium alone, as described in the testing for cross-tolerance section. For this part of the experiment, however, the culture was terminated 30, 60, or 120 min after the treatment, and the pooled samples from each culture bottle were stored frozen at -80°C. Embryonic and yolk sac tissues were stored and analyzed separately, since they have been shown to differ in the constitutive expression of hsp68 (Kothary et al., '87). Each group of embryo or yolk sac tissues was homogenized and proteins were solubilized in loading buffer and heated at 95°C for 3 min. Proteins were electro-

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phoresed in Phorcast gradient polyacrylamide gels (Amersham), with equal quantities of protein, as determined by a Biorad protein assay (Biorad, Mississauga, Ontario, Canada), loaded in each lane. After electrophoresis, the proteins were electroblotted to nitrocellulose. The membrane was blocked with 10% dried nonfat milk, and incubated overnight with the antihsp72 antibody. Bound antibody was visualized with a blotting detection kit containing an antimouse biotinylated secondary antibody, streptavidin-conjugated alkaline phosphatase and the substrates, 5-bromo-4chloro-3-indolyl phosphate and nitroblue tetrazolium (NBT) (Amersham). Two or three samples from each strain a t each time point were run in separate gels.

Changes in protein synthesis with heat and cadmium In order to determine whether there were any other differences between the two strains that could account for their responses to heat and cadmium, overall protein sysnthesis was examined. Three to four embryos were cultured in 1 ml serum and exposed to 43°C for 5 min, t o 1.75 pM cadmium, or to no treatment (control) as before; 1hr later, 50 pCi/ml of 35S-methionine (specific activity >1,000 Cilmmole) was added to the serum and embryos remained in the presence of the labelled amino acid for 60 min. At the end of this time, the pooled embryos from each culture bottle were rinsed in three changes of cold HBSS, separated into embryo and yolk sac portions, and stored frozen a t -80°C until use. Electrophoresis was carried out as before, but equal trichloroacetic acid (TCA)-precipitable counts were loaded in each lane (15,000 cpdlane). After electrophoresis, newly synthesized proteins were identified by fluorography: the gel was stained with Coomassie blue, soaked in Amplify (Amersham), dried under vacuum, and exposed to preflashed (Laskey and Mills, '75) Kodak XAR film at -80°C. Statistics Statistical analysis was carried out with the aid of CSS (Complete Statistical System, Statsoft, Inc., Tulsa, OK). Comparisons were made between the following pairs of groups: heat and control, cadmium and control, heaticadmium and heat, and heatlcadmium and cadmium. The frequency data

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Fig. 1. The effect of treatment with 5 min at 43"C, 1.75 pM cadmium, or a combination of the two on embryonic deaths and malformationsin BALBlc and SWV mice. (*, Cadmium alone was significantly different from control, P50.05;**, cadmium preceded by heat was significantly different from cadmium alone, Ps0.05).

(percentage dead or malformed) were analyzed by Fisher's exact test (Zar, '74). The morphological ratings, numbers of somites and other measurements were compared by analysis of variance (ANOVA) and the Newman-Keuls test (Zar, '74). The level of significance was P ~ 0 . 0 5 . RESULTS

Induction of tolerance to cadmium The heat pretreatment alone was found to have no embryolethal effect in BALB/c embryos but cadmium alone markedly increased the frequency of deaths compared to controls (Fig. 1, top). In the group of embryos exposed to heat before cadmium, the death rate was not significantly different

from cadmium alone. A similar set of responses was seen with malformations. Heat alone did not change the frequency compared to controls, and the increase in malformations that was caused by cadmium alone was not significantly diminished by the heat pretreatment. The most common malformations that were observed with the cadmium exposure (79% small forebrain, 71% microphthalmia, 71% small or abnormally shaped branchial arches) were similar to those previously observed in CD-1 mice (Kapron-Bras and Hales, '91). Very different responses to heat and cadmium were observed in SWV embryos from those observed in BALB/c embryos (Fig. 1, bottom). While heat alone was again seen to

GENETIC DIFFERENCES IN CROSS-TOLERANCE

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TABLJZ 1. Effects of heat (5 min 43°C) 0nd cadmium (1.75 pM) on the growth and development of BALBIc and SWV mouse embryos in uitro (mean f SEM) CrownYolk sac rump Head Prosencephalon Treatment NO.live No. Morphological diameter length length length embryos somites mnup score (mm) (mm) (mm) (mm) BALBic Control 31.3 t 0.4 19 47.2 0.8 4.49 * 0.07 3.78 2 0.08 1.82 f 0.07 0.41 2 0.02 Heat 46.7 f 0.7 19 31.0 -+ 0.3 4.52 0.08 3.66 2 0.08 1.76 2 0.06 0.41 ? 0.03 Cadmium 14 28.6 f 1.0' 41.4 ? 1.7' 3.37 2 0.15' 2.81 ? 0.17' 1.38 2 0.09' 0.28 ? 0.03' Heaticadmium 28.7 f 0.8' 42.1 ? 1.4' 3.43 i 0.11' 2.98 f 0.14' 1.42 ? 0.08' 0.28 ? 0.03' 15 SWV 29.9 -+ 0.4 Control 15 47.2 ? 0.6 4.59 ? 0.12 3.91 f 0.09 1.93 f 0.06 0.47 ? 0.02 Heat 47.4 2 0.7 17 30.5 ? 0.3 4.67 t 0.09 3.96 -t 0.08 1.99 ? 0.05 0.46 f 0.02 Cadmium 20 29.3 -t 0.4 45.0 & 0.9 4.01 ? 0.11' 3.53 f 0.13' 1.81 2 0.07 0.38 2 0.02l 21 Heaticadmium 30.2 2 0.8 47.3 * 1.1 4.35 f 0.133 3.90 5 0.113 2.04 t 0.08 0.49 ? 0.03"

*

'Significantly different from control by ANOVA (P

Genetic differences in heat-induced tolerance to cadmium in cultured mouse embryos are not correlated with changes in a 68-kD heat shock protein.

Heat-induced cross-tolerance to cadmium was investigated in two inbred strains of mice, BALB/c and SWV, using a whole embryo culture system. Embryos w...
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