Archives of Environmental Health: An International Journal
ISSN: 0003-9896 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/vzeh20
Hypoxia and Induced Mutations in Syrian (Golden) Hamsters Theodore H. Ingalls M. D. , Takamichi Shimada D. Sc. & Masaharu Yamamoto M. D. To cite this article: Theodore H. Ingalls M. D. , Takamichi Shimada D. Sc. & Masaharu Yamamoto M. D. (1976) Hypoxia and Induced Mutations in Syrian (Golden) Hamsters, Archives of Environmental Health: An International Journal, 31:3, 153-159, DOI: 10.1080/00039896.1976.10667210 To link to this article: http://dx.doi.org/10.1080/00039896.1976.10667210
Published online: 16 Apr 2013.
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11. College of American Pathologists, 1969. Systematized nomenclature of pathology. Chicago: College of American Pathologists. 12. Dontenwill, W.; Ranz, H.; and Mohr, U. 1960. Experimentene untersuchungen zur amyloidentstehung beim goldhamster. Beitr Path Anat 122:290-405. 13. Blaha, G. C. 1967. Histological features associated with amyloid deposits in organs of aged golden hamsters. Abstracted in Anat Rec 157:215. 14. Gleiser, C. A.; Van Hoosier, G. L.; Sheldon, W. G.; et a1. 1971. Amyloidosis and renal paramy10id in a closed hamster colony. Lab Anim Sci 21: 197-202. 15. Dontenwill, W.; Chevalier, H. J.; Harke, H. P.; et al. 1974. Die generalisierte amy loidose des syrischen goldhamsters bei chronischen berauchungsversuchen. Z Versuchstierkd 16: 75-84.
16. Dontenwill, W.; Chevalier, H. J.; Harke, H. P.; et al. 1973. Investigations on the effects of chronic cigarette-smoke inhalation in Syrian golden hamsters. J Natl Cancer Inst 51: 1781-1832. 17. Reckzeh, G.; Rucker, K.; Harke, H. P.; et al. 1969. Untersuchungen zur bestimmung der akuten und chronischen toxizitat von cigarettenrauch bei passiver berauchung von versuchstieren. Arzneim Forsch 19: 237-41. 18. Widdowson, E. M., and Kennedy, G. C. 1962. Rate of growth, mature weight and life-span. Proc R Soc Lond (Bioi) 156: 96-108. 19. Berg, B. N., and Simms, H. S. 1961. Nutrition and longevity in the rat: III. Food restriction beyond 800 days. J Nutr 74: 23-32. 20. Franklin, E. C., and Zucker-Franklin, D. 1972. Current concepts of amyloid. Adv Immunol15: 249-304.
Hypoxia and Induced Mutations in Syrian (Golden) Hamsters The Origin and Perpetuation of a Species
THEODORE H. INGALLS, M. D. Boston University School of Medicine and Framingham Union Hospital
TAKAMICHI SHIMADA, D. Sc. Yamagata University School of Medicine
MASAHARU YAMAMOTO, M. D. Niigata University School of Medicine
ABSTRACT We were able to identify anomalous chromosome patterns-monosomy, trisomy, and polyploidy-in metaphase plates prepared from embryos of Syrian hamsters (Mesocricetus auratus) whose mothers had been exposed to low-pressure hypoxia during the hours after copulation. These females were exposed in a low-pressure chamber for 4 hours at pressures equivalent to 30,000 feet of altitude before (estimated) OVUlation to create acid shifts of pH equilibrium in the milieu of newly released eggs during passage of sperm upward, during fertilization itself, and during early cleavage stages. The D group chromosomes were most frequently involved in both monosomy and hyperdiploidy. The easily identifiable X chromosome also appeared to be susceptible to aneuploid formation. Chromosomally abnormal patterns appeared most frequently among sibling embryos conceived by aging mothers exposed to lowpressure hypoxia. The significance of induced mutations is discussed.
THE CENTURY-OLD 1 ,2 and repeatedly confirmed observation 3 -6 of a maternal age-dependency of Down's syndrome has found a counterpart in recent years in the maternal age-dependency of other aneuploidies.7-9 Since, during the past several years, we had demonstrated by both in viV010 - 12 and in vitro13 experiments an etiological correlation between pH disturbances and aneuploid forma-
May/June 1976
tions, we undertook a new set of experiments to examine the hypothesiS that both factors (maternal age and pH disturbances) may act interdependently in the genesis of chromosomal anomalies in hamster embryos and, hence, in mutations that may determine the origin and perpetuation of a species.
153
manner but were kept at local atmospheric pressure (elevation, 500 feet) and at room temperature approximating 24°C. Both treated and control mothers were killed on the ninth day of gestation when the ovaries and uterine contents were surgically removed and promptly placed in Hanks' solution. The corpora lutea of pregnancy were counted under a dissecting microscope for comparison with the numbers of surviving embryos within the uterus. All embryos were put into Petri dishes with a glass pipet, immersed in Hanks' solution, and examined for gross abnormalities. Cytogenetic preparations were then made by the method of Dyban and Wroblewska. 14 The embryos were treated with a hypotonic solution of 0.9% sodium citrate for 15 to 30 minutes; they were fixed in a freshly prepared mixture of glacial acetic acid and methanol (in a volume-to-volume ratio of 1:3) for 2 hours; and after three changes of fixative, they were dissected into several pieces. The embryonic pieces were dissociated in a mixture of 50% lactic acid and glacial acetic acid (in a volume-to-volume ratio of 1:5) for 10 minutes, and spread on wet, cooled slides. Two drops of the fixative were immediately dropped on the slide and allowed to flow over the specimen. The slides were air dried and stained with Giemsa stain. More than twenty well-spread mitotic plates in metaphase were examined to detect aneuploidy and mosaicism. Karyotype analyses were carried out to identify any extra
Downloaded by [The University Of Melbourne Libraries] at 11:59 05 June 2016
Materials and Methods
The two groups of Syrian hamsters,Mesocricetus auratus, used in this study were composed of sexually mature, young female animals 3 to 6 months of age, and aging breeders approaching the end of their reproductive lives at 11 to 13 months of age. Both groups were kept under controlled conditions of temperature (24! 2°e) and air-conditioned humidity. Normal circadian rhythm of these animals had been reversed for at least 2 weeks by darkening of basement quarters during daylight hours from 6:00 a.m. to 6:00 p.m. and by illumination from 6:00 p.m. to 6:00 a.m. Selected female animals just coming into estrus were used; estrus is readily evidenced by the appearance of a sticky exudate, a manifestation that the animal will be receptive to copulation. Chosen females were placed with males at about 9 :00 a.m. and only those that had mated within 20 minutes were used as test animals. Beginning at about 9:30 a.m., these hamsters were then exposed for about 4 hours to low pressure equivalent to that found at 30,000 feet of altitude. 14 Thus, the immediate effects of this sustained exposure would be expected to occur well before ovulation during the final transition from the first meiotic metaphase to the second. The ensuing disturbance of pH equilibrium expectedly spans the process of polar body formation and may even disturb the sequences accompanying fertilization itself and mitotic processes of the zygote. Controls were allowed to copulate in a normal
Table 1. - Cytogenetic Findings on 9.-Day-Old Hamster Embryos From Four Groups of Mothers Cytogenetic Finding
Hypoxic* Young
Trisomy
Control
Old
0
Total
Young
Old
0
0
Monosomy D-
1
5
0
3
9
XO
0
3
0
0
3
0
2
9
0
0
2
0
5t
30
143
128
479
Mosaicism D+/N D-/N
5
Triploidy
1
Tetraploidy
0
6 2
Total embryos with anomalies
8t
17+
Normal embryos Male-female sex ratio Total embryos analyzed
111 131/100 119
5
97 137/100 114
127/100 143
121/100 133
509
*Low-p~essu.re
exposure at 30,000 feet for 4 hours (9:30 a.m. to 1 :30 p.m.). Estimated ovulation time, 1:00 to 4:00 p.m. of the day of estrous cycle (light, 6:00 p.m. to 6:00 a.m.).
t p < .05, compared with young controls. :j:P
ISSN: 0003-9896 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/vzeh20
Hypoxia and Induced Mutations in Syrian (Golden) Hamsters Theodore H. Ingalls M. D. , Takamichi Shimada D. Sc. & Masaharu Yamamoto M. D. To cite this article: Theodore H. Ingalls M. D. , Takamichi Shimada D. Sc. & Masaharu Yamamoto M. D. (1976) Hypoxia and Induced Mutations in Syrian (Golden) Hamsters, Archives of Environmental Health: An International Journal, 31:3, 153-159, DOI: 10.1080/00039896.1976.10667210 To link to this article: http://dx.doi.org/10.1080/00039896.1976.10667210
Published online: 16 Apr 2013.
Submit your article to this journal
Article views: 5
View related articles
Citing articles: 6 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=vzeh20 Download by: [The University Of Melbourne Libraries]
Date: 05 June 2016, At: 11:59
Downloaded by [The University Of Melbourne Libraries] at 11:59 05 June 2016
11. College of American Pathologists, 1969. Systematized nomenclature of pathology. Chicago: College of American Pathologists. 12. Dontenwill, W.; Ranz, H.; and Mohr, U. 1960. Experimentene untersuchungen zur amyloidentstehung beim goldhamster. Beitr Path Anat 122:290-405. 13. Blaha, G. C. 1967. Histological features associated with amyloid deposits in organs of aged golden hamsters. Abstracted in Anat Rec 157:215. 14. Gleiser, C. A.; Van Hoosier, G. L.; Sheldon, W. G.; et a1. 1971. Amyloidosis and renal paramy10id in a closed hamster colony. Lab Anim Sci 21: 197-202. 15. Dontenwill, W.; Chevalier, H. J.; Harke, H. P.; et al. 1974. Die generalisierte amy loidose des syrischen goldhamsters bei chronischen berauchungsversuchen. Z Versuchstierkd 16: 75-84.
16. Dontenwill, W.; Chevalier, H. J.; Harke, H. P.; et al. 1973. Investigations on the effects of chronic cigarette-smoke inhalation in Syrian golden hamsters. J Natl Cancer Inst 51: 1781-1832. 17. Reckzeh, G.; Rucker, K.; Harke, H. P.; et al. 1969. Untersuchungen zur bestimmung der akuten und chronischen toxizitat von cigarettenrauch bei passiver berauchung von versuchstieren. Arzneim Forsch 19: 237-41. 18. Widdowson, E. M., and Kennedy, G. C. 1962. Rate of growth, mature weight and life-span. Proc R Soc Lond (Bioi) 156: 96-108. 19. Berg, B. N., and Simms, H. S. 1961. Nutrition and longevity in the rat: III. Food restriction beyond 800 days. J Nutr 74: 23-32. 20. Franklin, E. C., and Zucker-Franklin, D. 1972. Current concepts of amyloid. Adv Immunol15: 249-304.
Hypoxia and Induced Mutations in Syrian (Golden) Hamsters The Origin and Perpetuation of a Species
THEODORE H. INGALLS, M. D. Boston University School of Medicine and Framingham Union Hospital
TAKAMICHI SHIMADA, D. Sc. Yamagata University School of Medicine
MASAHARU YAMAMOTO, M. D. Niigata University School of Medicine
ABSTRACT We were able to identify anomalous chromosome patterns-monosomy, trisomy, and polyploidy-in metaphase plates prepared from embryos of Syrian hamsters (Mesocricetus auratus) whose mothers had been exposed to low-pressure hypoxia during the hours after copulation. These females were exposed in a low-pressure chamber for 4 hours at pressures equivalent to 30,000 feet of altitude before (estimated) OVUlation to create acid shifts of pH equilibrium in the milieu of newly released eggs during passage of sperm upward, during fertilization itself, and during early cleavage stages. The D group chromosomes were most frequently involved in both monosomy and hyperdiploidy. The easily identifiable X chromosome also appeared to be susceptible to aneuploid formation. Chromosomally abnormal patterns appeared most frequently among sibling embryos conceived by aging mothers exposed to lowpressure hypoxia. The significance of induced mutations is discussed.
THE CENTURY-OLD 1 ,2 and repeatedly confirmed observation 3 -6 of a maternal age-dependency of Down's syndrome has found a counterpart in recent years in the maternal age-dependency of other aneuploidies.7-9 Since, during the past several years, we had demonstrated by both in viV010 - 12 and in vitro13 experiments an etiological correlation between pH disturbances and aneuploid forma-
May/June 1976
tions, we undertook a new set of experiments to examine the hypothesiS that both factors (maternal age and pH disturbances) may act interdependently in the genesis of chromosomal anomalies in hamster embryos and, hence, in mutations that may determine the origin and perpetuation of a species.
153
manner but were kept at local atmospheric pressure (elevation, 500 feet) and at room temperature approximating 24°C. Both treated and control mothers were killed on the ninth day of gestation when the ovaries and uterine contents were surgically removed and promptly placed in Hanks' solution. The corpora lutea of pregnancy were counted under a dissecting microscope for comparison with the numbers of surviving embryos within the uterus. All embryos were put into Petri dishes with a glass pipet, immersed in Hanks' solution, and examined for gross abnormalities. Cytogenetic preparations were then made by the method of Dyban and Wroblewska. 14 The embryos were treated with a hypotonic solution of 0.9% sodium citrate for 15 to 30 minutes; they were fixed in a freshly prepared mixture of glacial acetic acid and methanol (in a volume-to-volume ratio of 1:3) for 2 hours; and after three changes of fixative, they were dissected into several pieces. The embryonic pieces were dissociated in a mixture of 50% lactic acid and glacial acetic acid (in a volume-to-volume ratio of 1:5) for 10 minutes, and spread on wet, cooled slides. Two drops of the fixative were immediately dropped on the slide and allowed to flow over the specimen. The slides were air dried and stained with Giemsa stain. More than twenty well-spread mitotic plates in metaphase were examined to detect aneuploidy and mosaicism. Karyotype analyses were carried out to identify any extra
Downloaded by [The University Of Melbourne Libraries] at 11:59 05 June 2016
Materials and Methods
The two groups of Syrian hamsters,Mesocricetus auratus, used in this study were composed of sexually mature, young female animals 3 to 6 months of age, and aging breeders approaching the end of their reproductive lives at 11 to 13 months of age. Both groups were kept under controlled conditions of temperature (24! 2°e) and air-conditioned humidity. Normal circadian rhythm of these animals had been reversed for at least 2 weeks by darkening of basement quarters during daylight hours from 6:00 a.m. to 6:00 p.m. and by illumination from 6:00 p.m. to 6:00 a.m. Selected female animals just coming into estrus were used; estrus is readily evidenced by the appearance of a sticky exudate, a manifestation that the animal will be receptive to copulation. Chosen females were placed with males at about 9 :00 a.m. and only those that had mated within 20 minutes were used as test animals. Beginning at about 9:30 a.m., these hamsters were then exposed for about 4 hours to low pressure equivalent to that found at 30,000 feet of altitude. 14 Thus, the immediate effects of this sustained exposure would be expected to occur well before ovulation during the final transition from the first meiotic metaphase to the second. The ensuing disturbance of pH equilibrium expectedly spans the process of polar body formation and may even disturb the sequences accompanying fertilization itself and mitotic processes of the zygote. Controls were allowed to copulate in a normal
Table 1. - Cytogenetic Findings on 9.-Day-Old Hamster Embryos From Four Groups of Mothers Cytogenetic Finding
Hypoxic* Young
Trisomy
Control
Old
0
Total
Young
Old
0
0
Monosomy D-
1
5
0
3
9
XO
0
3
0
0
3
0
2
9
0
0
2
0
5t
30
143
128
479
Mosaicism D+/N D-/N
5
Triploidy
1
Tetraploidy
0
6 2
Total embryos with anomalies
8t
17+
Normal embryos Male-female sex ratio Total embryos analyzed
111 131/100 119
5
97 137/100 114
127/100 143
121/100 133
509
*Low-p~essu.re
exposure at 30,000 feet for 4 hours (9:30 a.m. to 1 :30 p.m.). Estimated ovulation time, 1:00 to 4:00 p.m. of the day of estrous cycle (light, 6:00 p.m. to 6:00 a.m.).
t p < .05, compared with young controls. :j:P
