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1944. Strain differences in cortisone and cortisol teratogenicity in mice
246
TERATOGENESIS
difficult to reconcile with reports from other workers, who had consistently reported that finely divided material did not produce local sarcomas. lit does not seem particularly surprising that local sarcomas developed around the shredded polyethylene contained in the gelatine capsules. After the dissolution of the capsule the shredded polyethylene must have remained as a compact lump, so that these implants would not, in fact, have differed greatly from the solid segments. Unfortunately one critical control group was omitted from the experiment. If tumours had been demonstrated in a control group in which the shredded material had been dispersed in the subcutaneous tissue, the authors would have been fully justified in questioning the work of other investigators and in postulating chemical reactivities to account for the tumours. We do not consider that the experimental data provided in this paper warrant such a challenge.] 1943. Spare a thought for the toxicologist!
Darlow, H. M., Simmons, D. J. C. & Roe, F. J. C. (1969). Hazards from experimental skin painting of carcinogens. A study based on use of a spore model. Archs envir. Hlth 18, 883. Preoccupied with the problems of protecting the consumers' interests (and the test animals' welfare toot), the toxicologist may perhaps be forgiven for overlooking an unexpected hazard that may confront him as a result of his handling of test materials. When the risk is obvious or immediate methods are soon devised to eliminate exposure to an offending material, but when the penalty of exposure takes many years to reveal itself, as may be the case with known or suspected chemical carcinogens, the necessity of avoiding the risk from the start may be overlooked. The authors cited above were particularly concerned with the safety of personnel coming into contact with animals exposed to chemical carcinogens. Spores of Bacillus globigii were employed as an experimental model to assess the likely environmental contamination resulting from skin painting with chemicals. It was found that spores persisted on the skin for as much as 16 days after a single application in an acetone suspension to the clipped dorsal skin of 30 mice, and during this period significant numbers of spores had dispersed into the atmosphere and bedding. In addition, re-clipping of the hair, changing the bedding or sweeping the floor added considerably to the atmospheric burden of spores. Further tests are contemplated using chemical carcinogens or closely-related noncarcinogenic materials to see whether such compounds are similarly disseminated, but the authors emphasize that until this work is completed the present findings should be interpreted with caution. [The authors are to be congratulated for directing attention to this problem and for their constructive approach to its investigation. It is too early to say whether the painting of skin with carcinogens, a commonly-used procedure, constitutes any carcinogenic hazard to the personnel involved under present-day conditions, but however small the possibility of risk, no effort should be spared to ensure that the working environment is not detrimental to health.]
TERATOGENESIS 1944. Strain differences in cortisone and cortisol teratogenicity in mice Loevy, Hannelore (1968). Cortisone-induced teratogenic effects in mice, Proc. Soc. exp. Biol. Med. 128, 841.
TERATOGENESIS
247
Levine, A., Yaffe, S. J. & Back, N. (1968). Maternal-fetal distribution of radioactive cortisol and its correlation with teratogenic effect. Proc. Soc. exp. Biol. Med. 129, 86. Cleft palate and harelip occur spontaneously in mice and their incidence varies widely according to strain. It is perhaps for this reason that mice are sometimes considered to be of doubtful value in teratological studies. In the first paper cited above, Loevy examines the incidence of these deformities in three strains of mice and some of their hybrids, both under normal conditions and after treatment with cortisone. The mice used were of the Strong A, C3H and BALB/c strains and their hybrids. Animals were injected subcutaneously on days 11-14 of pregnancy either with 1.25 mg cortisone/day, with equivalent doses of normal saline or with doses of 0.5 ml of a mixture containing sodium chloride, polysorbate 80 and sodium carboxymethylcellulose in the proportions used in the vehicle for the injected cortisone. A further control group received no injections. All animals were sacrificed on day 18 of pregnancy and the foetuses were dissected out, weighed and examined for cleft palate, harelip and other deformities. Spontaneous cleft palates were most frequent in the Strong A mice (9 70) but were rare in the C3H (< 1 70) and BALB/c (070) strains. A low incidence of spontaneously-occurring deformities of this type also occurred in the hybrids, notably in about 5 70 of foetuses derived from a backcross of C3H x Strong A females with pure Strong A males. After cortisone treatment, however, the incidence of cleft palate was significantly increased both in pure strain foetuses (10070 for Strong A, 36 70 for C3H and 62 70 for BALB/c ) and in hybrid foetuses. An interesting case occurred in foetuses derived from mating hybrid animals from Strong A and BALB/c parents with Strong A animals. The foetuses from untreated mothers showed a negligible rate of spontaneous malformation but exposure to cortisone during development caused an incidence of cleft palate of some 80 70 by day 18. A marked increase in this deformity was also seen in foetuses from cortisone-treated Strong A x C3H and Strong A parents. It has been suggested that the response of mice to cortisone treatment is largely dependent on maternal and foetal genotype, but this author notes that females of the same strain produced offspring with varying incidences of cleft palate, indicating that the ultimate response depended on the foetal genotype only. The specificity of cortisone in inducing cleft palate in mice is ably demonstrated by the failure of this drug to cause a significant increase in harelip, a deformity which is almost invariably present in mice with spontaneous cleft palate. The high incidence of cortisone-induced cleft palate in animals exhibiting a negligible spontaneous incidence suggests that the factors involved in its spontaneous development are unrelated to those determining susceptibility to this effect of cortisone. A great difference in strain susceptibility to cortisol has also been recognized since Warkany & Kalter (Plastic reconstr. Surg. 1962, 30, 628) reported a 100 70 incidence of cleft palate in A/Jax mice and only a 127o incidence in CBA mice after administration of a comparable dose of the steroid. Levine et al. (second paper, cited above) studied the foetal uptake of the drug and its rate of elimination in these two strains by administering subcutaneously on day 11 of pregnancy a single dose of 4.9/zg 14C-ring A-cortisol (with an activity of 40 t~c/mg) together with 2.5 mg of cold carrier. Mothers of each strain were sacrificed after 30, 60, 120 and 240 min and foetuses were individually homogenized and counted in a standard liquid scintillation counter. While it was concluded from the results that the placental transmissibility for the drugs was similar for the two strains, the radioactivity in the foetuses of the A/Jax strain at 30--240 min was very much higher than that in the CBA strain. This suggested that the isotope was removed more rapidly from the CBA strain and a higher level of the cortisol was retained by the A/Jax foetuses, which would be
248
TERATOGENESIS
expected also to include a much greater number of malformations. However in this experiment the foetuses were not examined for cleft palate or other deformities since the authors did not believe that a single dose of a teratogen, even at optimal levels, could produce abnormality. Although this type of experiment strongly indicates inherent differences in the metabolism of cortisol in these two strains, more extensive studies, in which the individual metabolites are isolated and characterized, are needed to provide a fuller explanation of these findings. [These experiments underline not only the importance of using adequate controls in teratological work but also the need for a good understanding of the genetic status of any mice that are to be employed.]
TERATOGENESIS
difficult to reconcile with reports from other workers, who had consistently reported that finely divided material did not produce local sarcomas. lit does not seem particularly surprising that local sarcomas developed around the shredded polyethylene contained in the gelatine capsules. After the dissolution of the capsule the shredded polyethylene must have remained as a compact lump, so that these implants would not, in fact, have differed greatly from the solid segments. Unfortunately one critical control group was omitted from the experiment. If tumours had been demonstrated in a control group in which the shredded material had been dispersed in the subcutaneous tissue, the authors would have been fully justified in questioning the work of other investigators and in postulating chemical reactivities to account for the tumours. We do not consider that the experimental data provided in this paper warrant such a challenge.] 1943. Spare a thought for the toxicologist!
Darlow, H. M., Simmons, D. J. C. & Roe, F. J. C. (1969). Hazards from experimental skin painting of carcinogens. A study based on use of a spore model. Archs envir. Hlth 18, 883. Preoccupied with the problems of protecting the consumers' interests (and the test animals' welfare toot), the toxicologist may perhaps be forgiven for overlooking an unexpected hazard that may confront him as a result of his handling of test materials. When the risk is obvious or immediate methods are soon devised to eliminate exposure to an offending material, but when the penalty of exposure takes many years to reveal itself, as may be the case with known or suspected chemical carcinogens, the necessity of avoiding the risk from the start may be overlooked. The authors cited above were particularly concerned with the safety of personnel coming into contact with animals exposed to chemical carcinogens. Spores of Bacillus globigii were employed as an experimental model to assess the likely environmental contamination resulting from skin painting with chemicals. It was found that spores persisted on the skin for as much as 16 days after a single application in an acetone suspension to the clipped dorsal skin of 30 mice, and during this period significant numbers of spores had dispersed into the atmosphere and bedding. In addition, re-clipping of the hair, changing the bedding or sweeping the floor added considerably to the atmospheric burden of spores. Further tests are contemplated using chemical carcinogens or closely-related noncarcinogenic materials to see whether such compounds are similarly disseminated, but the authors emphasize that until this work is completed the present findings should be interpreted with caution. [The authors are to be congratulated for directing attention to this problem and for their constructive approach to its investigation. It is too early to say whether the painting of skin with carcinogens, a commonly-used procedure, constitutes any carcinogenic hazard to the personnel involved under present-day conditions, but however small the possibility of risk, no effort should be spared to ensure that the working environment is not detrimental to health.]
TERATOGENESIS 1944. Strain differences in cortisone and cortisol teratogenicity in mice Loevy, Hannelore (1968). Cortisone-induced teratogenic effects in mice, Proc. Soc. exp. Biol. Med. 128, 841.
TERATOGENESIS
247
Levine, A., Yaffe, S. J. & Back, N. (1968). Maternal-fetal distribution of radioactive cortisol and its correlation with teratogenic effect. Proc. Soc. exp. Biol. Med. 129, 86. Cleft palate and harelip occur spontaneously in mice and their incidence varies widely according to strain. It is perhaps for this reason that mice are sometimes considered to be of doubtful value in teratological studies. In the first paper cited above, Loevy examines the incidence of these deformities in three strains of mice and some of their hybrids, both under normal conditions and after treatment with cortisone. The mice used were of the Strong A, C3H and BALB/c strains and their hybrids. Animals were injected subcutaneously on days 11-14 of pregnancy either with 1.25 mg cortisone/day, with equivalent doses of normal saline or with doses of 0.5 ml of a mixture containing sodium chloride, polysorbate 80 and sodium carboxymethylcellulose in the proportions used in the vehicle for the injected cortisone. A further control group received no injections. All animals were sacrificed on day 18 of pregnancy and the foetuses were dissected out, weighed and examined for cleft palate, harelip and other deformities. Spontaneous cleft palates were most frequent in the Strong A mice (9 70) but were rare in the C3H (< 1 70) and BALB/c (070) strains. A low incidence of spontaneously-occurring deformities of this type also occurred in the hybrids, notably in about 5 70 of foetuses derived from a backcross of C3H x Strong A females with pure Strong A males. After cortisone treatment, however, the incidence of cleft palate was significantly increased both in pure strain foetuses (10070 for Strong A, 36 70 for C3H and 62 70 for BALB/c ) and in hybrid foetuses. An interesting case occurred in foetuses derived from mating hybrid animals from Strong A and BALB/c parents with Strong A animals. The foetuses from untreated mothers showed a negligible rate of spontaneous malformation but exposure to cortisone during development caused an incidence of cleft palate of some 80 70 by day 18. A marked increase in this deformity was also seen in foetuses from cortisone-treated Strong A x C3H and Strong A parents. It has been suggested that the response of mice to cortisone treatment is largely dependent on maternal and foetal genotype, but this author notes that females of the same strain produced offspring with varying incidences of cleft palate, indicating that the ultimate response depended on the foetal genotype only. The specificity of cortisone in inducing cleft palate in mice is ably demonstrated by the failure of this drug to cause a significant increase in harelip, a deformity which is almost invariably present in mice with spontaneous cleft palate. The high incidence of cortisone-induced cleft palate in animals exhibiting a negligible spontaneous incidence suggests that the factors involved in its spontaneous development are unrelated to those determining susceptibility to this effect of cortisone. A great difference in strain susceptibility to cortisol has also been recognized since Warkany & Kalter (Plastic reconstr. Surg. 1962, 30, 628) reported a 100 70 incidence of cleft palate in A/Jax mice and only a 127o incidence in CBA mice after administration of a comparable dose of the steroid. Levine et al. (second paper, cited above) studied the foetal uptake of the drug and its rate of elimination in these two strains by administering subcutaneously on day 11 of pregnancy a single dose of 4.9/zg 14C-ring A-cortisol (with an activity of 40 t~c/mg) together with 2.5 mg of cold carrier. Mothers of each strain were sacrificed after 30, 60, 120 and 240 min and foetuses were individually homogenized and counted in a standard liquid scintillation counter. While it was concluded from the results that the placental transmissibility for the drugs was similar for the two strains, the radioactivity in the foetuses of the A/Jax strain at 30--240 min was very much higher than that in the CBA strain. This suggested that the isotope was removed more rapidly from the CBA strain and a higher level of the cortisol was retained by the A/Jax foetuses, which would be
248
TERATOGENESIS
expected also to include a much greater number of malformations. However in this experiment the foetuses were not examined for cleft palate or other deformities since the authors did not believe that a single dose of a teratogen, even at optimal levels, could produce abnormality. Although this type of experiment strongly indicates inherent differences in the metabolism of cortisol in these two strains, more extensive studies, in which the individual metabolites are isolated and characterized, are needed to provide a fuller explanation of these findings. [These experiments underline not only the importance of using adequate controls in teratological work but also the need for a good understanding of the genetic status of any mice that are to be employed.]