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Synergistic teratogenic effects of arsenic and hyperthermia in hamsters
ENVIKOSMEK~AL
14, 483-486 (19771
RtSEARCH
Synergistic
Teratogenic Hyperthermia
VERCIL H. FERM*
Effects of Arsenic in Hamsters AND
LAWRENCE
and
KrLHAMt
Received January 20, 1977 Similar malformations of the central nervous system including encephaloceles and exencephaly are induced by treatment of pregnant hamsters with sodium arsenate or hyperthermia on the eight day of gestation. These two treatments are synergistic in that combinations of minimal teratogenic levels of each cause a marked increase in fetal resorptions and the frequency and severity of developmental malformations as compared to separate treatments. The possible relationship of maternal hyperpyrexia and environmental teratogens to congenital malformations is discussed.
INTRODUCTION
The present investigation stems from two preceding ones; one of the effects of sodium arsenate (Ferm and Carpenter, 1968) and the other of hyperthermia(Kilham and Ferm, 1976) administered to pregnant hamsters on the eight day of gestation. Given in proper dosage, both agents induce a similar spectrum of developmental malformations. among which encephaloceles and exencephaly are the most striking. A question was whether these two agents, producing these same effects when given singly, would work synergistically if given together at or below the minimal teratogenic level. As explained below, each was given in amounts needed to induce malformations by itself. The results clearly indicated that the teratogenic potential of the two can be additive. Implications of this finding form the subject of a final discussion. MATERIALS
AND METHODS
Random-bred pregnant hamsters of the LVG strain were purchased from the Charles River Co. of North Wilmington, Massachusetts. The animals were divided, on gestation Day 8, into three groups, of which Groups I and III served as controls. Hamsters of Group I were first weighed and then after light ether anesthesia had body temperatures taken with an electric thermometer (Tele-thermometer, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio) and were given 0.5 ml of normal saline intraperitoneally (ip). After 1,2, or 3 hours, the animals were placed, for 50, 55, or 60 minutes, in a water-jacketed incubator set at 40°C. Body temperatures were taken again after removal from the incubator. Hamsters of Group II were treated in manner identical to those of Group I, except that they received sodium arsenate ip in a dose of 10 mg/kg, a dose known (Ferm and Carpenter, 1968: Fermet al., 1971) from previous experience to be near the minimal one needed to induce malformations in fetal hamsters. Hamsters of Group III were submitted to the same general procedures as those of the other two groups but, like the animals of Group II, received sodium arsenate in 483 CopyrIght @ 1977 by Academic Press. Inc. All rights of reproduction tn any form reserved.
ISSN
0013-9351
484
FERM
AND
KILHAM
doses of 10 mg/kg (ip) and were the only animals not submitted to the hyperthermia. In place of the incubator, they were kept in a dark cabinet at room temperature for an equivalent amount of time. An aim in all three groups was to see that all of the animals received equal amounts of handling. The three different exposure times (50,55, and 60 minutes) to hyperthermia and the three different intervals after the ip inoculations (1, 2, and 3 hours) made no difference in the results as far as we could determine. Some additional details of procedures used are given in a previous report on hyperthermia in pregnant hamsters (Kilham and Ferm, 1976). The animals were kept at all times in individual cages until killed with ether for examination on gestation Day 13. 14, or 15, the majority having been examined for malformations on Day 14. The rib anomalies were detectable at this time through the translucent skin. RESULTS
Results of experiments performed on Groups I, II, and III are summarized in Table 1 and Fig. 1. Taking the two control groups first, only four small encephaloceles were noted in 226 fetuses examined among the arsenic controls (Group III), and, among the 210 fetuses of the hyperthermia controls (Group I), seven encephaloceles and seven rib defects (fusions, bifurcations, stunting) were noted. None of these control fetuses developed exencephalies. As shown further in Table 1 and Fig. 1, the malformation rate increased considerably in mothers given arsenic and then submitted to hyperthermia (Group II). Thus, among 129 fetuses with malformations examined, 37, or 29%, had exencephalies, and 62 or 48%, had encephaloceles. As among the controls, rib defects were sometimes noted in association with head defects and, in other cases, as isolated abnormalities. Although the percentage of resorptions (127~) was greater in Group II than in the other groups, the increase was less striking than the increase in malformations. DISCUSSION
As recently described by Kilham and Ferm (1976), hyperthermia of 3 or 4°C can be a potent agent in induction of encephaloceles and exencephaly in fetal hamsters; TABLE EFFE~: I- OF !%)r~rrw
ARSENATE. ON EMBK~OSIC
1
HYPEK.I HERMIA, ASI) COMBIXED RESORPTION ANI) DEVELOPMEXT
SODILX ARSEN.~I-E-HVI~ER?.HF.R~~~~ IIV THE HAMSTER
Number of malformations in living fetuses” Group and treatment
Number of mothers
Number of gestation SXS
Number of fetal resorptions
Number of normal fetuses
Exencephaly
Encephalocele
Rib defects
I
Hyperthermia OlllY
20
230
16
210
0
7
7
II Arsenic and hyperthermia
31
375
4s
201
37
62
30
III Arsenic onIv
18
242
12
226
0
4
0
I’ For detads see text.
SYNERGISTIC
EFFECTS
OF As AND
HYPERTHERMIA
485
FIG. 1. Effect of sodium arsenate (10 mg/kg), hyperthermia, and combined sodium arsenatehyperthermia on embryonic resorption and developmental malformations in the golden hamster.
the report summarizes, in addition, the work of other investigators using heat in rats and guinea pigs. These same CNS as well as other malformations can be induced with sodium arsenate (Ferm et al., 1971). In both sets of experiments, those with hyperthermia and those with arsenic, the crucial point was treatment on gestation Day 8, which is a critical one in hamster embryogenesis. As shown in the present experiments, the combination of the two agents produces an additive teratogenic effect. An important question remaining is what are the possible mechanisms that may have underlain the synergism observed. Three hypotheses have occurred to us. The simplest of these is that heat accelerates chemical reactions and may have potentiated the arsenic in this way. This implies that the arsenic actually reaches the hamster embryo on Day 8 of gestation, a fact that has been demonstrated only recently by Hanlon and Ferm (unpublished data). A second hypothesis, based on these latter findings, is that hyperthermia might increase the permeability of the placenta and thus render a minimal dose of arsenate more teratogenic. A final hypothesis is that tissues with many cells in mitosis are especially sensitive to both heat and arsenic. In support of this are two reports: one of inhibition of mitotic activity and subsequent cell death in guinea pig fetuses on exposure of mothers to hyperthermia (Edwards, 1974) and the other on the action of arsenic in causing abnormal mitotic figures and a scattering of chromosomes in human marrow cells (Limarzi, 1943). If, as it would seem, both agents, the one physical and the other chemical, act on dividing cells in the embryo, then their joint effects in teratogenesis might be simply additive. In summary, it should be emphasized that the three hypotheses offered are not mutually exclusive and that actually all three mechanisms may have been operative in our experiments. The synergistic interaction of two teratogenic agents has been described in a number of other systems, including low doses of cortisone with hypocaloric diets (Kalter, 1960), 3-acetylpyridine with 6-aminonicotinamide (Landauer and Clark, 1962), and lead with cadmium (Ferm, 1969). The use of hyperthermia with a highly
486
FERM
AND
KILHAM
specific chemical teratogen, arsenic, presents new possibilities for investigations. A further point, and an important one, is that maternal hyperpyexia arising from infection or other cause during critical stages of embryonic development might become a threat if it acts in association with other teratogenic agents, chemical or otherwise, present in the environment or latent in body tissues. ACKNOWLEDGMENTS This work was supported by NIH Grants ES-00697 (V.F.) and HD-07775 and Research Cancer Program Award I-K-6-CA 22652 from the National Cancer Institute (L.K.).
REFERENCES Edwards, M. J. (1974). The effects of hyperthermia on pregnancy and prenatal development. E.rp. Embtyol. Teratol. 1, 90-133. Ferm, V. H. (1%9). The synteratognic effect of lead and cadmium. Experientiu 25, 5657. Ferm, V. H., and Carpenter, S. J. (1968). Malformations induced by sodium arsenate. J. Reprod. Fert. 17, 199-201. Ferm, V. H., Saxon, A., and Smith, B. M. (1971). The teratogenic profile of sodium arsenate in the golden hamster. Arch. Environ. Health 22, 557-560. Kalter, H. (l%O). Teratogenic action of a hypocaloric diet and small doses of cortisone. Proc. Sot. Exp. Biol. Med. 104, SllX-520. Kilham, L., and Ferm, V. H. ( 1976). Exencephaly in fetal hamsters following exposure to hyperthermia. Teratology 14, 323-326. Landauer, W., and Clark, E. M. (1%2). The interaction in teratogenic activity of two niacin analogs 3-acetylpryidine and 6-aminonicotinamide. J. Exp. Zoo/. 151, 253-258. Limarzi, L. R. (1943). The effect of arsenic (Fowler’s solution) on erythropoiesis. Amer. J. Med. Sci. 206, 339-347.
14, 483-486 (19771
RtSEARCH
Synergistic
Teratogenic Hyperthermia
VERCIL H. FERM*
Effects of Arsenic in Hamsters AND
LAWRENCE
and
KrLHAMt
Received January 20, 1977 Similar malformations of the central nervous system including encephaloceles and exencephaly are induced by treatment of pregnant hamsters with sodium arsenate or hyperthermia on the eight day of gestation. These two treatments are synergistic in that combinations of minimal teratogenic levels of each cause a marked increase in fetal resorptions and the frequency and severity of developmental malformations as compared to separate treatments. The possible relationship of maternal hyperpyrexia and environmental teratogens to congenital malformations is discussed.
INTRODUCTION
The present investigation stems from two preceding ones; one of the effects of sodium arsenate (Ferm and Carpenter, 1968) and the other of hyperthermia(Kilham and Ferm, 1976) administered to pregnant hamsters on the eight day of gestation. Given in proper dosage, both agents induce a similar spectrum of developmental malformations. among which encephaloceles and exencephaly are the most striking. A question was whether these two agents, producing these same effects when given singly, would work synergistically if given together at or below the minimal teratogenic level. As explained below, each was given in amounts needed to induce malformations by itself. The results clearly indicated that the teratogenic potential of the two can be additive. Implications of this finding form the subject of a final discussion. MATERIALS
AND METHODS
Random-bred pregnant hamsters of the LVG strain were purchased from the Charles River Co. of North Wilmington, Massachusetts. The animals were divided, on gestation Day 8, into three groups, of which Groups I and III served as controls. Hamsters of Group I were first weighed and then after light ether anesthesia had body temperatures taken with an electric thermometer (Tele-thermometer, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio) and were given 0.5 ml of normal saline intraperitoneally (ip). After 1,2, or 3 hours, the animals were placed, for 50, 55, or 60 minutes, in a water-jacketed incubator set at 40°C. Body temperatures were taken again after removal from the incubator. Hamsters of Group II were treated in manner identical to those of Group I, except that they received sodium arsenate ip in a dose of 10 mg/kg, a dose known (Ferm and Carpenter, 1968: Fermet al., 1971) from previous experience to be near the minimal one needed to induce malformations in fetal hamsters. Hamsters of Group III were submitted to the same general procedures as those of the other two groups but, like the animals of Group II, received sodium arsenate in 483 CopyrIght @ 1977 by Academic Press. Inc. All rights of reproduction tn any form reserved.
ISSN
0013-9351
484
FERM
AND
KILHAM
doses of 10 mg/kg (ip) and were the only animals not submitted to the hyperthermia. In place of the incubator, they were kept in a dark cabinet at room temperature for an equivalent amount of time. An aim in all three groups was to see that all of the animals received equal amounts of handling. The three different exposure times (50,55, and 60 minutes) to hyperthermia and the three different intervals after the ip inoculations (1, 2, and 3 hours) made no difference in the results as far as we could determine. Some additional details of procedures used are given in a previous report on hyperthermia in pregnant hamsters (Kilham and Ferm, 1976). The animals were kept at all times in individual cages until killed with ether for examination on gestation Day 13. 14, or 15, the majority having been examined for malformations on Day 14. The rib anomalies were detectable at this time through the translucent skin. RESULTS
Results of experiments performed on Groups I, II, and III are summarized in Table 1 and Fig. 1. Taking the two control groups first, only four small encephaloceles were noted in 226 fetuses examined among the arsenic controls (Group III), and, among the 210 fetuses of the hyperthermia controls (Group I), seven encephaloceles and seven rib defects (fusions, bifurcations, stunting) were noted. None of these control fetuses developed exencephalies. As shown further in Table 1 and Fig. 1, the malformation rate increased considerably in mothers given arsenic and then submitted to hyperthermia (Group II). Thus, among 129 fetuses with malformations examined, 37, or 29%, had exencephalies, and 62 or 48%, had encephaloceles. As among the controls, rib defects were sometimes noted in association with head defects and, in other cases, as isolated abnormalities. Although the percentage of resorptions (127~) was greater in Group II than in the other groups, the increase was less striking than the increase in malformations. DISCUSSION
As recently described by Kilham and Ferm (1976), hyperthermia of 3 or 4°C can be a potent agent in induction of encephaloceles and exencephaly in fetal hamsters; TABLE EFFE~: I- OF !%)r~rrw
ARSENATE. ON EMBK~OSIC
1
HYPEK.I HERMIA, ASI) COMBIXED RESORPTION ANI) DEVELOPMEXT
SODILX ARSEN.~I-E-HVI~ER?.HF.R~~~~ IIV THE HAMSTER
Number of malformations in living fetuses” Group and treatment
Number of mothers
Number of gestation SXS
Number of fetal resorptions
Number of normal fetuses
Exencephaly
Encephalocele
Rib defects
I
Hyperthermia OlllY
20
230
16
210
0
7
7
II Arsenic and hyperthermia
31
375
4s
201
37
62
30
III Arsenic onIv
18
242
12
226
0
4
0
I’ For detads see text.
SYNERGISTIC
EFFECTS
OF As AND
HYPERTHERMIA
485
FIG. 1. Effect of sodium arsenate (10 mg/kg), hyperthermia, and combined sodium arsenatehyperthermia on embryonic resorption and developmental malformations in the golden hamster.
the report summarizes, in addition, the work of other investigators using heat in rats and guinea pigs. These same CNS as well as other malformations can be induced with sodium arsenate (Ferm et al., 1971). In both sets of experiments, those with hyperthermia and those with arsenic, the crucial point was treatment on gestation Day 8, which is a critical one in hamster embryogenesis. As shown in the present experiments, the combination of the two agents produces an additive teratogenic effect. An important question remaining is what are the possible mechanisms that may have underlain the synergism observed. Three hypotheses have occurred to us. The simplest of these is that heat accelerates chemical reactions and may have potentiated the arsenic in this way. This implies that the arsenic actually reaches the hamster embryo on Day 8 of gestation, a fact that has been demonstrated only recently by Hanlon and Ferm (unpublished data). A second hypothesis, based on these latter findings, is that hyperthermia might increase the permeability of the placenta and thus render a minimal dose of arsenate more teratogenic. A final hypothesis is that tissues with many cells in mitosis are especially sensitive to both heat and arsenic. In support of this are two reports: one of inhibition of mitotic activity and subsequent cell death in guinea pig fetuses on exposure of mothers to hyperthermia (Edwards, 1974) and the other on the action of arsenic in causing abnormal mitotic figures and a scattering of chromosomes in human marrow cells (Limarzi, 1943). If, as it would seem, both agents, the one physical and the other chemical, act on dividing cells in the embryo, then their joint effects in teratogenesis might be simply additive. In summary, it should be emphasized that the three hypotheses offered are not mutually exclusive and that actually all three mechanisms may have been operative in our experiments. The synergistic interaction of two teratogenic agents has been described in a number of other systems, including low doses of cortisone with hypocaloric diets (Kalter, 1960), 3-acetylpyridine with 6-aminonicotinamide (Landauer and Clark, 1962), and lead with cadmium (Ferm, 1969). The use of hyperthermia with a highly
486
FERM
AND
KILHAM
specific chemical teratogen, arsenic, presents new possibilities for investigations. A further point, and an important one, is that maternal hyperpyexia arising from infection or other cause during critical stages of embryonic development might become a threat if it acts in association with other teratogenic agents, chemical or otherwise, present in the environment or latent in body tissues. ACKNOWLEDGMENTS This work was supported by NIH Grants ES-00697 (V.F.) and HD-07775 and Research Cancer Program Award I-K-6-CA 22652 from the National Cancer Institute (L.K.).
REFERENCES Edwards, M. J. (1974). The effects of hyperthermia on pregnancy and prenatal development. E.rp. Embtyol. Teratol. 1, 90-133. Ferm, V. H. (1%9). The synteratognic effect of lead and cadmium. Experientiu 25, 5657. Ferm, V. H., and Carpenter, S. J. (1968). Malformations induced by sodium arsenate. J. Reprod. Fert. 17, 199-201. Ferm, V. H., Saxon, A., and Smith, B. M. (1971). The teratogenic profile of sodium arsenate in the golden hamster. Arch. Environ. Health 22, 557-560. Kalter, H. (l%O). Teratogenic action of a hypocaloric diet and small doses of cortisone. Proc. Sot. Exp. Biol. Med. 104, SllX-520. Kilham, L., and Ferm, V. H. ( 1976). Exencephaly in fetal hamsters following exposure to hyperthermia. Teratology 14, 323-326. Landauer, W., and Clark, E. M. (1%2). The interaction in teratogenic activity of two niacin analogs 3-acetylpryidine and 6-aminonicotinamide. J. Exp. Zoo/. 151, 253-258. Limarzi, L. R. (1943). The effect of arsenic (Fowler’s solution) on erythropoiesis. Amer. J. Med. Sci. 206, 339-347.