TERATOLOGY 43:83-94 (1991)
Heat-Shock Induced Tolerance to the Embryotoxic Effects of Hyperthermia and Cadmium in Mouse Embryos in Vitro CAROLYN M. KAPRON-BRAS AND BARBARA F. HALES Department of Pharmacology and Therapeutics and Centre for the Study Reproduction, McGill University, Montreal, Quebec, Canada H3G 1 Y6
of
ABSTRACT
Mammalian embryos growing in vitro are harmed by short elevations in the culture temperature. However, a relatively mild hyperthermic exposure can induce thermotolerance, a transient state of resistance to the effects of a subsequent heat exposure. The present study examines the induction of tolerance t o heat and cross-tolerance to another teratogen, cadmium, in day 8 CD-1 mouse embryos in vitro. The ability of a mild heat pretreatment (5 min a t 43°C) to partially protect embryos against an embryotoxic heat exposure (20 min at 43°C) was demonstrated. The frequency of death was reduced from 43% to 20%, abnormal branchial arches from 44% to 13.2%,and retarded turning from 22% to 5% in pretreated embryos. Other malformations, such as small forebrains and microphthalmia, were not affected, and the rate of exencephaly was significantly increased. The same heat pretreatment (5 min at 43°C) was also found to reduce the damaging effects of a subsequent exposure to 1.75 FM cadmium. In the absence of pretreatment, cadmium caused 55%embryo deaths and 87% malformations, but prior heat exposure caused significant reductions in these frequencies to 29% and 55%. The total morphological score was higher in the pretreated group, as were the measurements of the yolk sac diameter, crown-rump length, and head length. Thus, embryos that have developed resistance to hyperthermia are also partially protected against the harmful effects of a second teratogen, cadmium. The response of the embryo to elevated temperatures may be involved in the development of tolerance to a variety of stresses.
Bournias-Vardiabasis, '82; Li et al., '83;Abernethy et al., '89), as well as a variety of cells growing in culture (Henle, '87). Thermotolerance may be induced by stresses other than hyperthermia, such as ethanol (Boon-Niermeijer et al., '88) and 2,4-dinitrophenol (Boon-Niermeijer et al., '87) in freshwater snails; puromycin (Lee and Dewey, '87) and glucocorticoids (Fisher et al., '86) in Chinese hamster ovary (CHO) cells; and sodium arsenite, cadmium chloride, ethanol, and hypoxia in Chinese hamster fibroblasts (Li et al., '82). '87). In contrast t o the effects of a toxic expoThe mechanisms of the thermotolerance sure, a nonteratogenic heat pretreatment response are not yet fully understood. Expohas been shown to protect rat embryos in sures to elevated temperatures are known vitro against the damaging effects of a subsequent hyperthermic exposure (Mirkes, '87; Walsh et al., '87).The acquisition of tolReceived April 5, 1990; accepted August 15, 1990. erance to heat has also been documented reprint requests to Dr. Barbara F. Hales, Department for numerous other organisms, including of Address Pharmacology and Therapeutics, McGill University, Monplants, insects, and adult mice (Buzin and treal, Quebec, Canada H3G 1Y6.
Exposure of embryos to experimentally elevated temperatures during organogenesis has long been known to be embryotoxic (see review by Edwards, '86). When hyperthermia occurs in rodents in vivo, the central nervous system (CNS) is particularly sensitive t o damage (Webster and Edwards, '84; Finnell et al., '86; Shiota, '88); in vitro exposure of rat embryos leads to malformations, such as microcephaly, microphthalmia, and pericardial edema (Cockroft and New, '75, '78; Mirkes, '85; Walsh et al.,
0 1991 WILEY-LISS, INC.
84
C.M. KAPRON-BRAS AND B.F. HALES
to alter transcription and translation in mammalian cells. Heat causes a transient depression in overall protein synthesis and at the same time an increase in the synthesis of a small set of proteins known as the heat shock proteins (Lindquist and Craig, '88). These generally fall into three major groups, whose molecular masses are in the neighborhood of 90, 70, and 27 kD. In response to elevated temperatures, rat embryos (Mirkes, '87; Walsh et al., '87) and differentiating cultures of Drosophila larval cells (Buzin and Bournias-Vardiabasis, '84) have been shown to produce heat-shock proteins. It appears that at least some of the proteins induced by hyperthermia are essential for protection against heat (Li et al., '82; Riabowol et al., '88; Landry et al., '89). However, instances of the induction of thermotolerance without protein synthesis suggest that other factors may be important as well (Boon-Niermeijer et al., '87). In the search of the mechanisms of the embryonic thermotolerance response, it would be of interest to determine whether heat exposure plays a role in preventing the embryotoxicity of other teratogens. There is some evidence that heat can induce tolerance to the cytotoxic effects of drugs such as Adriamycin (Li et al., '82) and bleomycin (Hahn et al., '89) in cultured cells and to the inhibition of differentiation by diphenylhydantoin and coumarin in Drosophila embryonic cells in culture (Buzin and BourniasVardiabasis, '82). To test for possible cross-tolerance in cultured mouse embryos, cadmium was chosen as a model teratogen. Cadmium has previously been shown to be toxic to mouse embryos both in vivo (Wolkowski, '74; Warner et al., '83) and in vitro (Warner et al., '84; Nakashima and Fujiki, '87), causing embryonic death and malformations, such as exencephaly, abnormal brains, and retarded turning. The addition of cadmium to cultured cells (Li et al., '82) and t o primary Drosophila embryonic cultures (BourniasVardiabasis and Buzin, '86) has been shown to induce the synthesis of heat-shock proteins. As well, cadmium has been shown t o induce tolerance to heat in CHO cells (Li et al., '82). However, the ability of heat to provide cross-tolerance to cadmium has, to the best of our knowledge, never been determined. Thus, the present study examines how a hyperthermic exposure alters the state of
the embryo by testing the hypothesis that a heat treatment that induces thermotolerance in an embryo will also cause it to be resistant to another teratogen. MATERIALS AND METHODS
Mice CD-1 mice, purchased from Charles River Canada, Inc. (St. Constant, Quebec), were housed under a 12-h lightdark cycle and supplied with Purina Mouse Chow and tap water ad libitum. Timed pregnant females were obtained by placing 1-4 females with each male for 3 h in the morning. Mice with a vaginal plug at the end of this time period were considered to be in day 0 of pregnancy. Embryo culture Mouse embryos were cultured using the method of New ('78) for rat embryos, with some modifications. At 10 AM on day 8 of gestation, pregnant females were killed by cervical dislocation. The decidua were dissected from the uterus in Hank's Balanced Salt Solution (HBSS) (Gibco, Burlington, Ontario), under aseptic conditions. Decidual tissue and Reichert's membrane were removed and the visceral yolk sac and the ectoplacental cone left intact. Embryos chosen for culture had 4-8 somites. Three embryos were placed into each 60-ml glass bottle containing 5.4 ml filter-sterilized (0.45-kM pore diameter filters) medium, made up of 10% Tyrode's saline (Gibco), and 90% immediately centrifuged, heat-inactivated rat serum. Just before culture, 10 Ulml penicillin G and 10 +g/ml streptomycin (Gibco)were added. The bottles were gassed with 5% 02-5% C0290% N, at the start of culture and 20% 025% co2-75% N2 at 18 and 26 h of culture. They were placed in a 37°C incubator and rotated at 30 rpm. The cultures were terminated after a total of 42 h, and the embryos were examined for survival, as indicated by the presence of a heartbeat and yolk sac circulation. The yolk sac diameter, crownrump length, and head length of live embryos were subsequently measured using an eyepiece micrometer. The live embryos were checked for any external malformations and then evaluated by the scoring system of Brown and Fabro ('81). Under this system, individual morphological features are given a numerical score, between 1 and 5, that corresponds to a given stage of differentiation. The sum of the individual scores gives
85
HEAT-INDUCED TOLERANCE TO CADMIUM
a total morphological score that is indicative of the stage of development of each embryo.
Selection of heat and cadmium treatments The bottles containing the embryos were equilibrated at 37°C for 2 h. The heattreated bottles were then placed upright in a 43°C waterbath for 5 , 10, 20, or 30 min. The medium in the bottle reached the designated temperature within 2 min and remained within 0.2% of that temperature for the treatment time. Control embryo bottles remained upright in the 37°C incubator. In a separate experiment, cadmium was added to the medium 2.5 h after the start of culture in the form of cadmium chloride in distilled water. The final concentrations of cadmium were 1.0, 1.75, and 2.25 pM; the metal remained in the medium throughout the culture period. Distilled water alone was added to control bottles. Induction of thermotolerance The bottles containing the embryos were equilibrated a t 37°C for 2 h and were then divided into two groups. One group was placed upright in a 43°C waterbath for 5 min, a treatment that had been found not to be embryotoxic. This group is referred to as the pretreated embryos. The nonpretreated group consisted of control embryos that were left in upright bottles in the 37°C incubator for the 5-min period. To test for the induction of thermotolerance, all embryos were returned to 37°C for 30 min after the heat pretreatment. Then, one-half of the pretreated group and onehalf of the nonpretreated group were placed in the 43°C waterbath for 20 min, an exposure that had been found to be embryotoxic. Thus, four groups of embryos were included in the experiment. The first group remained at 37°C throughout the culture period (designated O/O). The second group was exposed only to the pretreatment of 43°C for 5 min 6/01. The third group was heated a t 43°C for 20 min with no pretreatment (0/20). The fourth group was pretreated a t 43°C for 5 min and subsequently exposed to the same temperature for 20 rnin (5/20). Induction of tolerance to cadmium A similar experimental design was used to test for the induction of tolerance to cadmium. The embryos were pretreated at 43°C for 5 min or left a t 37"C,as described above.
Y
0
5
10
20
30
TIME AT 43OC (rnin)
Fig. 1. Effect of exposure to 5, 10, 20, or 30 min at 43°C on the frequencies of embryolethality and malformations. *Significantly different from 0 rnin control by Fisher's exact test; FY0.05.
Thirty minutes later, cadmium chloride in 100 pl distilled water was added to the culture medium in one-half of the pretreated and nonpretreated groups. The other bottles received distilled water alone. The final concentration of cadmium was 1.75 p M , which had been found to be embryotoxic. Four groups of embryos made up this part of the study. The first group remained at 37°C throughout the culture period and was not exposed to cadmium (O/O). The second group received only the pretreatment (5/0). The third group received 1.75 pM cadmium, with no pretreatment (04.75). The fourth group was pretreated a t 43°C for 5 min and then exposed to 1.75 pM cadmium (31.75).
Statistics Statistical analysis was carried out with the aid of a CSS (Complete Statistical System) computer program (Statsoft, Inc., Tulsa, OK). The frequency data (percentage dead, percentage malformed) were analysed by Fisher's exact test (Zar, '74) or the G-test for independence (Sokal and Rohlf, '69). The morphological ratings, numbers of somites, yolk sac diameters, crown-rump lengths, and head lengths were compared by analysis of variance (ANOVA) and by the Newman-Keuls test (Zar, '74). In all cases, a value of P50.05 was considered significant. RESULTS
Selection of heat and cadmium treatments The exposure of day 8 mouse embryos to short periods of elevated culture temperature was found to be embryotoxic (Fig. 1). A temperature of43"C for 5 or 10 min did not
86
C.M. KAPRON-BRAS AND B.F. HALES
0
1.00
1.75
2.25
CADMIUM CONCENTRATION (pM)
Fig. 2. Effect of exposure to 1.00, 1.75, or 2.25 pM cadmium on the frequencies of embryolethality and malformations. *Significantly different from 0 pM control by Fisher’s exact test; P
Heat-Shock Induced Tolerance to the Embryotoxic Effects of Hyperthermia and Cadmium in Mouse Embryos in Vitro CAROLYN M. KAPRON-BRAS AND BARBARA F. HALES Department of Pharmacology and Therapeutics and Centre for the Study Reproduction, McGill University, Montreal, Quebec, Canada H3G 1 Y6
of
ABSTRACT
Mammalian embryos growing in vitro are harmed by short elevations in the culture temperature. However, a relatively mild hyperthermic exposure can induce thermotolerance, a transient state of resistance to the effects of a subsequent heat exposure. The present study examines the induction of tolerance t o heat and cross-tolerance to another teratogen, cadmium, in day 8 CD-1 mouse embryos in vitro. The ability of a mild heat pretreatment (5 min a t 43°C) to partially protect embryos against an embryotoxic heat exposure (20 min at 43°C) was demonstrated. The frequency of death was reduced from 43% to 20%, abnormal branchial arches from 44% to 13.2%,and retarded turning from 22% to 5% in pretreated embryos. Other malformations, such as small forebrains and microphthalmia, were not affected, and the rate of exencephaly was significantly increased. The same heat pretreatment (5 min at 43°C) was also found to reduce the damaging effects of a subsequent exposure to 1.75 FM cadmium. In the absence of pretreatment, cadmium caused 55%embryo deaths and 87% malformations, but prior heat exposure caused significant reductions in these frequencies to 29% and 55%. The total morphological score was higher in the pretreated group, as were the measurements of the yolk sac diameter, crown-rump length, and head length. Thus, embryos that have developed resistance to hyperthermia are also partially protected against the harmful effects of a second teratogen, cadmium. The response of the embryo to elevated temperatures may be involved in the development of tolerance to a variety of stresses.
Bournias-Vardiabasis, '82; Li et al., '83;Abernethy et al., '89), as well as a variety of cells growing in culture (Henle, '87). Thermotolerance may be induced by stresses other than hyperthermia, such as ethanol (Boon-Niermeijer et al., '88) and 2,4-dinitrophenol (Boon-Niermeijer et al., '87) in freshwater snails; puromycin (Lee and Dewey, '87) and glucocorticoids (Fisher et al., '86) in Chinese hamster ovary (CHO) cells; and sodium arsenite, cadmium chloride, ethanol, and hypoxia in Chinese hamster fibroblasts (Li et al., '82). '87). In contrast t o the effects of a toxic expoThe mechanisms of the thermotolerance sure, a nonteratogenic heat pretreatment response are not yet fully understood. Expohas been shown to protect rat embryos in sures to elevated temperatures are known vitro against the damaging effects of a subsequent hyperthermic exposure (Mirkes, '87; Walsh et al., '87).The acquisition of tolReceived April 5, 1990; accepted August 15, 1990. erance to heat has also been documented reprint requests to Dr. Barbara F. Hales, Department for numerous other organisms, including of Address Pharmacology and Therapeutics, McGill University, Monplants, insects, and adult mice (Buzin and treal, Quebec, Canada H3G 1Y6.
Exposure of embryos to experimentally elevated temperatures during organogenesis has long been known to be embryotoxic (see review by Edwards, '86). When hyperthermia occurs in rodents in vivo, the central nervous system (CNS) is particularly sensitive t o damage (Webster and Edwards, '84; Finnell et al., '86; Shiota, '88); in vitro exposure of rat embryos leads to malformations, such as microcephaly, microphthalmia, and pericardial edema (Cockroft and New, '75, '78; Mirkes, '85; Walsh et al.,
0 1991 WILEY-LISS, INC.
84
C.M. KAPRON-BRAS AND B.F. HALES
to alter transcription and translation in mammalian cells. Heat causes a transient depression in overall protein synthesis and at the same time an increase in the synthesis of a small set of proteins known as the heat shock proteins (Lindquist and Craig, '88). These generally fall into three major groups, whose molecular masses are in the neighborhood of 90, 70, and 27 kD. In response to elevated temperatures, rat embryos (Mirkes, '87; Walsh et al., '87) and differentiating cultures of Drosophila larval cells (Buzin and Bournias-Vardiabasis, '84) have been shown to produce heat-shock proteins. It appears that at least some of the proteins induced by hyperthermia are essential for protection against heat (Li et al., '82; Riabowol et al., '88; Landry et al., '89). However, instances of the induction of thermotolerance without protein synthesis suggest that other factors may be important as well (Boon-Niermeijer et al., '87). In the search of the mechanisms of the embryonic thermotolerance response, it would be of interest to determine whether heat exposure plays a role in preventing the embryotoxicity of other teratogens. There is some evidence that heat can induce tolerance to the cytotoxic effects of drugs such as Adriamycin (Li et al., '82) and bleomycin (Hahn et al., '89) in cultured cells and to the inhibition of differentiation by diphenylhydantoin and coumarin in Drosophila embryonic cells in culture (Buzin and BourniasVardiabasis, '82). To test for possible cross-tolerance in cultured mouse embryos, cadmium was chosen as a model teratogen. Cadmium has previously been shown to be toxic to mouse embryos both in vivo (Wolkowski, '74; Warner et al., '83) and in vitro (Warner et al., '84; Nakashima and Fujiki, '87), causing embryonic death and malformations, such as exencephaly, abnormal brains, and retarded turning. The addition of cadmium to cultured cells (Li et al., '82) and t o primary Drosophila embryonic cultures (BourniasVardiabasis and Buzin, '86) has been shown to induce the synthesis of heat-shock proteins. As well, cadmium has been shown t o induce tolerance to heat in CHO cells (Li et al., '82). However, the ability of heat to provide cross-tolerance to cadmium has, to the best of our knowledge, never been determined. Thus, the present study examines how a hyperthermic exposure alters the state of
the embryo by testing the hypothesis that a heat treatment that induces thermotolerance in an embryo will also cause it to be resistant to another teratogen. MATERIALS AND METHODS
Mice CD-1 mice, purchased from Charles River Canada, Inc. (St. Constant, Quebec), were housed under a 12-h lightdark cycle and supplied with Purina Mouse Chow and tap water ad libitum. Timed pregnant females were obtained by placing 1-4 females with each male for 3 h in the morning. Mice with a vaginal plug at the end of this time period were considered to be in day 0 of pregnancy. Embryo culture Mouse embryos were cultured using the method of New ('78) for rat embryos, with some modifications. At 10 AM on day 8 of gestation, pregnant females were killed by cervical dislocation. The decidua were dissected from the uterus in Hank's Balanced Salt Solution (HBSS) (Gibco, Burlington, Ontario), under aseptic conditions. Decidual tissue and Reichert's membrane were removed and the visceral yolk sac and the ectoplacental cone left intact. Embryos chosen for culture had 4-8 somites. Three embryos were placed into each 60-ml glass bottle containing 5.4 ml filter-sterilized (0.45-kM pore diameter filters) medium, made up of 10% Tyrode's saline (Gibco), and 90% immediately centrifuged, heat-inactivated rat serum. Just before culture, 10 Ulml penicillin G and 10 +g/ml streptomycin (Gibco)were added. The bottles were gassed with 5% 02-5% C0290% N, at the start of culture and 20% 025% co2-75% N2 at 18 and 26 h of culture. They were placed in a 37°C incubator and rotated at 30 rpm. The cultures were terminated after a total of 42 h, and the embryos were examined for survival, as indicated by the presence of a heartbeat and yolk sac circulation. The yolk sac diameter, crownrump length, and head length of live embryos were subsequently measured using an eyepiece micrometer. The live embryos were checked for any external malformations and then evaluated by the scoring system of Brown and Fabro ('81). Under this system, individual morphological features are given a numerical score, between 1 and 5, that corresponds to a given stage of differentiation. The sum of the individual scores gives
85
HEAT-INDUCED TOLERANCE TO CADMIUM
a total morphological score that is indicative of the stage of development of each embryo.
Selection of heat and cadmium treatments The bottles containing the embryos were equilibrated at 37°C for 2 h. The heattreated bottles were then placed upright in a 43°C waterbath for 5 , 10, 20, or 30 min. The medium in the bottle reached the designated temperature within 2 min and remained within 0.2% of that temperature for the treatment time. Control embryo bottles remained upright in the 37°C incubator. In a separate experiment, cadmium was added to the medium 2.5 h after the start of culture in the form of cadmium chloride in distilled water. The final concentrations of cadmium were 1.0, 1.75, and 2.25 pM; the metal remained in the medium throughout the culture period. Distilled water alone was added to control bottles. Induction of thermotolerance The bottles containing the embryos were equilibrated a t 37°C for 2 h and were then divided into two groups. One group was placed upright in a 43°C waterbath for 5 min, a treatment that had been found not to be embryotoxic. This group is referred to as the pretreated embryos. The nonpretreated group consisted of control embryos that were left in upright bottles in the 37°C incubator for the 5-min period. To test for the induction of thermotolerance, all embryos were returned to 37°C for 30 min after the heat pretreatment. Then, one-half of the pretreated group and onehalf of the nonpretreated group were placed in the 43°C waterbath for 20 min, an exposure that had been found to be embryotoxic. Thus, four groups of embryos were included in the experiment. The first group remained at 37°C throughout the culture period (designated O/O). The second group was exposed only to the pretreatment of 43°C for 5 min 6/01. The third group was heated a t 43°C for 20 min with no pretreatment (0/20). The fourth group was pretreated a t 43°C for 5 min and subsequently exposed to the same temperature for 20 rnin (5/20). Induction of tolerance to cadmium A similar experimental design was used to test for the induction of tolerance to cadmium. The embryos were pretreated at 43°C for 5 min or left a t 37"C,as described above.
Y
0
5
10
20
30
TIME AT 43OC (rnin)
Fig. 1. Effect of exposure to 5, 10, 20, or 30 min at 43°C on the frequencies of embryolethality and malformations. *Significantly different from 0 rnin control by Fisher's exact test; FY0.05.
Thirty minutes later, cadmium chloride in 100 pl distilled water was added to the culture medium in one-half of the pretreated and nonpretreated groups. The other bottles received distilled water alone. The final concentration of cadmium was 1.75 p M , which had been found to be embryotoxic. Four groups of embryos made up this part of the study. The first group remained at 37°C throughout the culture period and was not exposed to cadmium (O/O). The second group received only the pretreatment (5/0). The third group received 1.75 pM cadmium, with no pretreatment (04.75). The fourth group was pretreated a t 43°C for 5 min and then exposed to 1.75 pM cadmium (31.75).
Statistics Statistical analysis was carried out with the aid of a CSS (Complete Statistical System) computer program (Statsoft, Inc., Tulsa, OK). The frequency data (percentage dead, percentage malformed) were analysed by Fisher's exact test (Zar, '74) or the G-test for independence (Sokal and Rohlf, '69). The morphological ratings, numbers of somites, yolk sac diameters, crown-rump lengths, and head lengths were compared by analysis of variance (ANOVA) and by the Newman-Keuls test (Zar, '74). In all cases, a value of P50.05 was considered significant. RESULTS
Selection of heat and cadmium treatments The exposure of day 8 mouse embryos to short periods of elevated culture temperature was found to be embryotoxic (Fig. 1). A temperature of43"C for 5 or 10 min did not
86
C.M. KAPRON-BRAS AND B.F. HALES
0
1.00
1.75
2.25
CADMIUM CONCENTRATION (pM)
Fig. 2. Effect of exposure to 1.00, 1.75, or 2.25 pM cadmium on the frequencies of embryolethality and malformations. *Significantly different from 0 pM control by Fisher’s exact test; P
