Vol. 26, No. 8, Augost 1975 Printed in U.S.A.
FERTILITY AND STERILITY Copyright~ 1975 The American Fertility Society
COMPARISON OF CHROMOSOME BREAKAGES IN LYMPHOCYTES AND FIBROBLASTS FROM CONTROL WOMEN AND WOMEN TAKING ORAL CONTRACEPTIVES* L. GAYLE LITTLEFIELD, PH.D.,
AND
JOHN B. MAILHES, PH.D.t
Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830
Several investigators have reported increased frequencies of chromosome breakages in lymphocyte cultures from women taking oral contraceptives (0C),t· 3 whereas other studies have shown no increase in aberration frequencies in cultures from pill users. 4 • 5 Our laboratory recently completed a longitudinal study of chromosome breakages in 849 lymphocyte cultures from 54 normal women, including "control" women, pregnant women, and women taking OC. 6 Although we observed a statistically significant increase in average breakages in a series of cultures from the group of OC users, we did not observe higher breakages in cultures from all women taking OC nor in all cultures from any single pill user. Likewise, we observed considerable variability in the frequency of "spontaneous" breakages in lymphocyte cultures from the control women, both within cultures from the same person and among cultures from different women. The variability in aberration frequencies in the control cultures, combined with sporadic instances of very high breakages (>20%) in some preparations suggested that the lymphocyte may be very susceptible to chromosome damage induced by a variety of extraneous factors.
This observation raised the question, does exposure to agents such as OC, which apparently results in a slight increase in average breakages in lymphocyte chromosomes, also result in chromosome damage in other somatic cells? To answer this question, we initiated a series of studies to determine the in vivo and in vitro effects of synthetic hormones on a second type of human cell, the skin fibroblast. By culturing peripheral blood lymphocytes and skin fibroblasts from groups of control women and pill users, we hoped to determine (1) whether there were differences in breakage frequencies between the two types of cells, (2) whether the frequency of chromosome breakages or of cytogenetically aberrant stem-lines of cells was higher in fibroblasts from women taking OC, and (3) whether chromosome breakages were induced in fibroblasts exposed to serum from OC users. In this communication we present our findings on chromosome aberration frequencies in these lymphocyte and fibroblast cultures. Detailed descriptions of our stem-line data will be presented elsewhere. 7 MATERIALS AND METHODS
During an 18-month period we obtained a minimum of four sets of blood samples Received October 8, 1974. *Supported by the Energy Research and Develop- and skin biopsies from each of five women ment Administration and National Institute of who were taking combination type OC and Child Health and Human Development Interagency from five women who had never taken Agreement 40-152-68. synthetic hormones. All women were t Present address: Department ofPediatrics, Louisbetween the ages of 20 and 40, in good iana State University Medical Center, Shreveport, La. 71101. health, and had no history of congenital 828
Vol. 26, No.8
EFFECT OF ORAL CONTRACEPTIVES ON CHROMOSOMES
abnormalities or medical defects. At the time each blood sample and skin biopsy were obtained, each woman filled out a health sheet describing recent illnesses and incidental medications taken, and recorded the date of her last menstrual cycle. We set up the following series of cultures: Lymphocyte Preparations. We initiated two lymphocyte cultures from each blood sample: one culture was supplemented with autologous serum and the other with commercially processed fetal calf serum (FCS) (final concentration of serum, 20% ). The cells were harvested after 3 days' incubation at 37" C, and slides were made for cytogenetic evaluation. The culture methods, fixation, and staining procedures have been described in detail elsewhere.8 Fibroblast Preparations. On the same day that venous blood samples were drawn for lymphocyte cultures, we obtained a full-thickness skin biopsy from each woman. Primary fibroblast cultures were initiated as previously described 9 and were maintained using standard tissue culture procedures. When sufficient fibroblasts had been accumulated for the cytogenetic studies, we exposed replicate subcultures of dividing cells to fresh medium containing the following types of serum: (1) 20% FCS, (2) 20% autologous serum, (3) 20% homologous serum (from a control woman), and (4) 20% homologous serum (from an OC user). After approximately 24 hours of exposure to the test sera, we processed the cultures for cytogenetic analyses. The lymphocyte and fibroblast cultures were given code numbers so that the five persons scoring the slides would not be aware of the hormonal status of the tissue donor. Scoring Procedures. In evaluating these cultures for chromosomal aberrations, we had to take into account that, in contrast to lymphocytes which normally progress through only one to three divisions in 72-hour cultures, 10 fibroblasts derived
829
from primary tissue must proceed through several in vitro divisions before a sufficient number of cells is available for cytogenetic study. Those fibroblasts which suffered lethal type cytogenetic injuries in vivo would be eliminated during the culture period, whereas cells bearing nonlethal type chromosomal rearrangements might become established in culture. We evaluated all fibroblast cultures both for chromosomal aberrations that may have been induced by exposure to various sera and for stem-lines of abnormal cells that may have evolved as a consequence of in vivo or in vitro injury to chromosomes. We attempted to score for chromosomal abnormalities 100 well stained, well spread metaphases from each lymphocyte and fibroblast culture. Metaphases with good morphology were counted, and those having 45 or 46 centromeres were analyzed for chromosomal breakages and rearrangements. The frequencies of single chromatid and iso-chromatid breaks, chromatid exchanges, deletions, translocations, inversions, dicentrics, and rings were determined for each culture. If a specific chromosomal aberration (e.g., reciprocal translocation) was noted in three or more cells in the same replicate series of fibroblast cultures, we scored the aberrant metaphases as a stem-line and did not include the lesion in breakage tabulations. RESULTS
We obtained analyzable metaphases from 20 sets of blood and fibroblast cultures from the 5 control women, and from 17 sets of cultures from the 5 pill users. The culture data and cytogenetic findings are shown in Table 1. To determine whether there were differences in chromosome aberration frequencies in the various lymphocyte and fibroblast cultures, we calculated the average number (plus or minus standard error) of metaphases having chromosome breakages in the various sets of cultures. The most frequently observed lesions in all
830
August 1975
LITI'LEFIELD AND MAILHES
TABLE 1. Chromosome Aberrations in Lymphocytes and Fibroblasts from Control Women and Women Taking Oral Contraceptives
Cell type Control women Lymphocytes Lymphocytes Fibroblasts Fibroblasts Fibroblasts Fibroblasts OC usersd Lymphocytes Lymphocytes Fibroblasts Fibroblasts Fibroblasts Fibroblasts
cells
No. of scored
Avera!hr~~o~~;:::tbfe~kS:s with
treatment«
No. of cultures analyzedh
Autologous FCS Autologous FCS Homologous Homologous OC
19 (1) 18 (2) 19 (1) 14 (6) 17 (3) 15 (5)
1713 1391 1528 1002 1394 1113
5.62 5.84 6.51 5.57 5.82 5.84
± ± ± ± ± ±
1.17 3.06 1.61 0.89 0.89 1.46
3.04 3.06 3.90 2.96 3.63 2.79
± ± ± ± ± ±
1.03 1.00 1.39 0.92 0.83 0.99
Autologous FCS Autologous FCS Homologous Homologous OC
17 (0) 14 (3) 15 (2) 13 (4) 15 (2) 15 (2)
1510 770 1447 947 1403 1368
4.99 7.10 4.60 6.23 5.09 4.88
± ± ± ± ± ±
1.00 1.39 0.89 1.27 0.70 0.69
2.43 3.83 1.86 2.52 2.15 2.48
± ± ± ± ± ±
0.81 1.38 0.44 1.04 0.28 0.54
Serum
c~~~~~~i! b~~~~s
ct~!;:~~l~ b~::f.~
aFibroblast cultures were exposed to various test sera (final concentration, 20%) for approximately 24 hours immediately prior to harvest. hThe number of failures (cultures with fewer than 20 metaphases analyzed) is indicated in parentheses. Data from these were not included in breakage tabulations. cAll culture data were adjusted to number of abnormal cells per 100 cells; values ± standard error. dFour women were taking Ortho-Novum 1/50 (Ortho Pharmaceutical Corporation, Raritan, N.J.), and one woman was taking Ovulen-21 (Searle & Co., San Juan, P.R.).
groups of cultures were chromatid and chromosome breaks. Complex lesions requiring two or more breaks for their formation (i.e., chromatid exchanges, dicentrics, translocations, etc.) were seen infrequently. The frequency of metaphases having damaged chromosomes appeared to be similar in all groups of cultures, and statistical comparisons of the means (2tailed t-test) verified that there were no differences in breakages within series of lymphocyte and fibroblast cultures treated with the various types of sera, or among lymphocyte and fibroblast cultures from the control women, compared with cultures from women taking OC. Thus, in these experiments, fibroblasts exposed to autologous serum or FCS had the same frequency of breakages as lymphocytes cultured in media containing the same serum, and no increase in breakages could be detected in fibroblasts exposed to serum from OC users. Likewise, there was no difference in breakages in lymphocytes and fibroblasts from OC users, compared
with lymphocytes and fibroblasts in the control women. Evaluation ofStem-Lines. We identified subpopulations of cells having consistent cytogenetic abnormalities in seven series of fibroblast cultures. Five of the stemlines were observed in fibroblasts from the control women and two in fibroblasts from OC users. We detected clones of cells having consistent structural abnormalities (translocations, inversions, dicentrics) as well as a cell line monosomic for a D group chromosome and a pseudodiploid cell line monosomic for a no. 3 chromosome and trisomic for a C group chromosome. The various stem-lines were observed in cultures ranging from 41 to 144 days old and comprised varying percentages of the cell populations, ranging from less than 1% to almost 100%. DISCUSSION
By culturing blood lymphocytes and skin fibroblasts from a group of control women and OC users, we attempted to
Vol. 26, No.8
EFFECT OF ORAL CONTRACEPTIVES ON CHROMOSOMES
determine whether these synthetic hormones caused increased cytogenetic aberrations in human somatic cells other than lymphocytes. We chose to study fibroblasts because these cells are relatively easy to culture and are the only somatic cells other than lymphocytes that can be obtained with a minimum of trauma from human volunteers. We realized at the outset that there were some problems is using this cell type for estimates of aberrations induced in vivo. Because of the loss of cells bearing some types of aberrations and the possible evolution of stem-lines of other abnormal cells, it follows that the aberrations observed in fibroblasts cultured in vitro for a period of several weeks would not necessarily reflect the in vivo condition of this population of cells. Thus, in an effort to determine the effect of OC on chromosomes, we not only analyzed breakages in standard cultures exposed to FCS but also exposed replicate fibroblast cultures to autologous serum, homologous serum, and homologous serum from OC users. From the series of replicate cultures we hoped to learn whether the frequency of breakages or stable rearrangements was higher in the standard cultures from the OC users, whether fibroblasts from women who had taken OC would show increased breakages or abnormal growth when re-exposed to serum containing synthetic hormonal constituents, and whether serum from OC users contained substances that could induce breakages in fibroblasts from women who had not taken oral contraceptives. Results of cytogenetic analyses of these fibroblast cultures showed no difference in chromosome breakages in cells from OC users cultured in FCS, compared with control cells cultured in the same serum, and we could not detect any evidence of breakage induced by OC serum in either the control fibroblasts or fibroblasts from OC users. Likewise, we found no evidence
831
for increased frequency of cytogenetically abnormal stem-lines in fibroblasts from the women taking OC. In fact, we observed 5 aberrant clones of cells in 19 primary cultures derived from skin from the control women, and only 2 aberrant clones in 17 primary cultures from the OC users. We do not know whether the 7 abnormal cell lines observed in these cultures arose from cells that suffered injuries in vivo or whether the clones evolved as a result of an injury incurred during the period of culture, but the lack of observation of a substantial number of chromosomally aberrant stem-lines in the 17 sets of cultures from the OC users certainly suggests that fibroblasts from these women are no more predisposed to the evolution of aberrant clones than are fibroblasts from women who have not taken synthetic hormones. In the present series of experiments we did not observe an increase in chromosome breakages in lymphocyte cultures from five women taking OC, compared with breakages in cultures from five women who had never taken synthetic hormones. This findings is not unexpected since our larger survey study of aberrations in lymphocytes from pill users 6 had demonstrated considerable variability in breakages in consecutive cultures from the same individual, and among cultures from different persons. Likewise, we had previously found that average breakages in series of cultures from some OC users were not different from breakages in cultures from some control women. Because of the variability in breakages among cultures from the same individual, we obtained four separate sets of tissue samples from each woman for the evaluation of chromosome lesions. However, sl.nce we obtained blood from only five women in each group, it is possible that our sample size was not large enough to detect the minor increases in breakages that we had observed in the larger group of OC users.
832
LITTLEFIELD AND MAll..HES
SUMMARY
To determine whether exposure to synthetic hormones resulted in increased chromosome damage in cells other than lymphocytes, we evaluated series of lymphocyte and fibroblast cultures from five control women and five women taking oral contraceptives (OC). The results of this study showed: {1) no difference in chromosome breakages between lymphocytes and fibroblasts; (2) no differences in breakages in replicate fibroblast cultures exposed to fetal calf serum, autologous serum, homologous serum, or homologous serum from OC users; (3) no difference in breakages between lymphocyte and fibroblast cultures from OC users, compared with lymphocyte and fibroblast cultures from control women; and (4) no increase in the frequency of cytogenetically aberrant stem-lines in fibroblast cultures from women taking OC. These findings suggest that synthetic hormones do not cause increased chromosome breakages in fibroblasts from women taking OC. Acknowledgment. The authors gratefully acknowledge the technical assistance of Mr. Barry Lazar, Mr. Eugene Joiner, and Ms. Lydia Corrill, and express appreciation to Mr. F. L. Miller, who conducted the statistical analyses.
2.
3.
4. 5.
6.
7.
8.
9.
REFERENCES 1. Goh KO: Chromosomal breaks in women taking
birth control pills. In Research Report, 1967Medical Division, Oak Ridge Associated Uni-
10 ·
August 1975
versities. Oak Ridge, Tenn, US AEC Report ORAU-106, 1968, p 97 McQuarrie HG, Scott CD, Ellsworth HS, Harris JW, Stone RA: Cytogenetic studies on women using oral contraceptives and their progeny. Am J Obstet Gynecol108:659, 1970 Badr FM, Badr RS, lbrakin A, Shaaban HA: Effect of oral and injectable contraceptives on the human chromosomes. In Fourth International Congress of Human Genetics, Paris, 6-11 September 1971, Abstracts of Papers Presented (International Congress Series 233). Amsterdam, Excerpta Medica, 1971, p 20 Shapiro LR, Graves ZR, Hirschhorn K: Oral contraceptives and in vivo cytogenetic studies. Obstet Gynecol 39:190, 1972 de Gutierrez AC, Lisker R: Longitudinal study of the effects of oral contraceptives on human chromosomes. Ann Genet (Paris) 16:259, 1973 Littlefield LG, Lever WE, Miller FL, Goh KO: Chromosome-breakage studies in lymphocytes from normal women, pregnant women, and women taking oral contraceptives. Am J Obstet Gynecol 121:976, 1975 Littlefield LG, Mailhes J: Observations of de novo clones of cytogenetically aberrant cells in primary fibroblast cell strains from phenotypically normal women. Am J Hum Genet 27:190, 1975 Littlefield LG, Goh KO: Cytogenetic studies in control men and women. 1. Variations in aberration frequencies in 29,709 metaphases from 305 cultures obtained over a three-year period. Cytogenet Cell Genet 12:17, 1973 Mailhes JB, Lazar BS, Littlefield LG: A reliable procedure for the in vitro culture of human fibroblasts. In Research Report, 1971-Medical Division, Oak Ridge Associated Universities. Oak Ridge, Tenn, US AEC Report ORAU-122, 1973, p 177 Sasaki M, Norman A: Proliferation of human lymphocytes in culture. Nature 210:913, 1966
FERTILITY AND STERILITY Copyright~ 1975 The American Fertility Society
COMPARISON OF CHROMOSOME BREAKAGES IN LYMPHOCYTES AND FIBROBLASTS FROM CONTROL WOMEN AND WOMEN TAKING ORAL CONTRACEPTIVES* L. GAYLE LITTLEFIELD, PH.D.,
AND
JOHN B. MAILHES, PH.D.t
Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830
Several investigators have reported increased frequencies of chromosome breakages in lymphocyte cultures from women taking oral contraceptives (0C),t· 3 whereas other studies have shown no increase in aberration frequencies in cultures from pill users. 4 • 5 Our laboratory recently completed a longitudinal study of chromosome breakages in 849 lymphocyte cultures from 54 normal women, including "control" women, pregnant women, and women taking OC. 6 Although we observed a statistically significant increase in average breakages in a series of cultures from the group of OC users, we did not observe higher breakages in cultures from all women taking OC nor in all cultures from any single pill user. Likewise, we observed considerable variability in the frequency of "spontaneous" breakages in lymphocyte cultures from the control women, both within cultures from the same person and among cultures from different women. The variability in aberration frequencies in the control cultures, combined with sporadic instances of very high breakages (>20%) in some preparations suggested that the lymphocyte may be very susceptible to chromosome damage induced by a variety of extraneous factors.
This observation raised the question, does exposure to agents such as OC, which apparently results in a slight increase in average breakages in lymphocyte chromosomes, also result in chromosome damage in other somatic cells? To answer this question, we initiated a series of studies to determine the in vivo and in vitro effects of synthetic hormones on a second type of human cell, the skin fibroblast. By culturing peripheral blood lymphocytes and skin fibroblasts from groups of control women and pill users, we hoped to determine (1) whether there were differences in breakage frequencies between the two types of cells, (2) whether the frequency of chromosome breakages or of cytogenetically aberrant stem-lines of cells was higher in fibroblasts from women taking OC, and (3) whether chromosome breakages were induced in fibroblasts exposed to serum from OC users. In this communication we present our findings on chromosome aberration frequencies in these lymphocyte and fibroblast cultures. Detailed descriptions of our stem-line data will be presented elsewhere. 7 MATERIALS AND METHODS
During an 18-month period we obtained a minimum of four sets of blood samples Received October 8, 1974. *Supported by the Energy Research and Develop- and skin biopsies from each of five women ment Administration and National Institute of who were taking combination type OC and Child Health and Human Development Interagency from five women who had never taken Agreement 40-152-68. synthetic hormones. All women were t Present address: Department ofPediatrics, Louisbetween the ages of 20 and 40, in good iana State University Medical Center, Shreveport, La. 71101. health, and had no history of congenital 828
Vol. 26, No.8
EFFECT OF ORAL CONTRACEPTIVES ON CHROMOSOMES
abnormalities or medical defects. At the time each blood sample and skin biopsy were obtained, each woman filled out a health sheet describing recent illnesses and incidental medications taken, and recorded the date of her last menstrual cycle. We set up the following series of cultures: Lymphocyte Preparations. We initiated two lymphocyte cultures from each blood sample: one culture was supplemented with autologous serum and the other with commercially processed fetal calf serum (FCS) (final concentration of serum, 20% ). The cells were harvested after 3 days' incubation at 37" C, and slides were made for cytogenetic evaluation. The culture methods, fixation, and staining procedures have been described in detail elsewhere.8 Fibroblast Preparations. On the same day that venous blood samples were drawn for lymphocyte cultures, we obtained a full-thickness skin biopsy from each woman. Primary fibroblast cultures were initiated as previously described 9 and were maintained using standard tissue culture procedures. When sufficient fibroblasts had been accumulated for the cytogenetic studies, we exposed replicate subcultures of dividing cells to fresh medium containing the following types of serum: (1) 20% FCS, (2) 20% autologous serum, (3) 20% homologous serum (from a control woman), and (4) 20% homologous serum (from an OC user). After approximately 24 hours of exposure to the test sera, we processed the cultures for cytogenetic analyses. The lymphocyte and fibroblast cultures were given code numbers so that the five persons scoring the slides would not be aware of the hormonal status of the tissue donor. Scoring Procedures. In evaluating these cultures for chromosomal aberrations, we had to take into account that, in contrast to lymphocytes which normally progress through only one to three divisions in 72-hour cultures, 10 fibroblasts derived
829
from primary tissue must proceed through several in vitro divisions before a sufficient number of cells is available for cytogenetic study. Those fibroblasts which suffered lethal type cytogenetic injuries in vivo would be eliminated during the culture period, whereas cells bearing nonlethal type chromosomal rearrangements might become established in culture. We evaluated all fibroblast cultures both for chromosomal aberrations that may have been induced by exposure to various sera and for stem-lines of abnormal cells that may have evolved as a consequence of in vivo or in vitro injury to chromosomes. We attempted to score for chromosomal abnormalities 100 well stained, well spread metaphases from each lymphocyte and fibroblast culture. Metaphases with good morphology were counted, and those having 45 or 46 centromeres were analyzed for chromosomal breakages and rearrangements. The frequencies of single chromatid and iso-chromatid breaks, chromatid exchanges, deletions, translocations, inversions, dicentrics, and rings were determined for each culture. If a specific chromosomal aberration (e.g., reciprocal translocation) was noted in three or more cells in the same replicate series of fibroblast cultures, we scored the aberrant metaphases as a stem-line and did not include the lesion in breakage tabulations. RESULTS
We obtained analyzable metaphases from 20 sets of blood and fibroblast cultures from the 5 control women, and from 17 sets of cultures from the 5 pill users. The culture data and cytogenetic findings are shown in Table 1. To determine whether there were differences in chromosome aberration frequencies in the various lymphocyte and fibroblast cultures, we calculated the average number (plus or minus standard error) of metaphases having chromosome breakages in the various sets of cultures. The most frequently observed lesions in all
830
August 1975
LITI'LEFIELD AND MAILHES
TABLE 1. Chromosome Aberrations in Lymphocytes and Fibroblasts from Control Women and Women Taking Oral Contraceptives
Cell type Control women Lymphocytes Lymphocytes Fibroblasts Fibroblasts Fibroblasts Fibroblasts OC usersd Lymphocytes Lymphocytes Fibroblasts Fibroblasts Fibroblasts Fibroblasts
cells
No. of scored
Avera!hr~~o~~;:::tbfe~kS:s with
treatment«
No. of cultures analyzedh
Autologous FCS Autologous FCS Homologous Homologous OC
19 (1) 18 (2) 19 (1) 14 (6) 17 (3) 15 (5)
1713 1391 1528 1002 1394 1113
5.62 5.84 6.51 5.57 5.82 5.84
± ± ± ± ± ±
1.17 3.06 1.61 0.89 0.89 1.46
3.04 3.06 3.90 2.96 3.63 2.79
± ± ± ± ± ±
1.03 1.00 1.39 0.92 0.83 0.99
Autologous FCS Autologous FCS Homologous Homologous OC
17 (0) 14 (3) 15 (2) 13 (4) 15 (2) 15 (2)
1510 770 1447 947 1403 1368
4.99 7.10 4.60 6.23 5.09 4.88
± ± ± ± ± ±
1.00 1.39 0.89 1.27 0.70 0.69
2.43 3.83 1.86 2.52 2.15 2.48
± ± ± ± ± ±
0.81 1.38 0.44 1.04 0.28 0.54
Serum
c~~~~~~i! b~~~~s
ct~!;:~~l~ b~::f.~
aFibroblast cultures were exposed to various test sera (final concentration, 20%) for approximately 24 hours immediately prior to harvest. hThe number of failures (cultures with fewer than 20 metaphases analyzed) is indicated in parentheses. Data from these were not included in breakage tabulations. cAll culture data were adjusted to number of abnormal cells per 100 cells; values ± standard error. dFour women were taking Ortho-Novum 1/50 (Ortho Pharmaceutical Corporation, Raritan, N.J.), and one woman was taking Ovulen-21 (Searle & Co., San Juan, P.R.).
groups of cultures were chromatid and chromosome breaks. Complex lesions requiring two or more breaks for their formation (i.e., chromatid exchanges, dicentrics, translocations, etc.) were seen infrequently. The frequency of metaphases having damaged chromosomes appeared to be similar in all groups of cultures, and statistical comparisons of the means (2tailed t-test) verified that there were no differences in breakages within series of lymphocyte and fibroblast cultures treated with the various types of sera, or among lymphocyte and fibroblast cultures from the control women, compared with cultures from women taking OC. Thus, in these experiments, fibroblasts exposed to autologous serum or FCS had the same frequency of breakages as lymphocytes cultured in media containing the same serum, and no increase in breakages could be detected in fibroblasts exposed to serum from OC users. Likewise, there was no difference in breakages in lymphocytes and fibroblasts from OC users, compared
with lymphocytes and fibroblasts in the control women. Evaluation ofStem-Lines. We identified subpopulations of cells having consistent cytogenetic abnormalities in seven series of fibroblast cultures. Five of the stemlines were observed in fibroblasts from the control women and two in fibroblasts from OC users. We detected clones of cells having consistent structural abnormalities (translocations, inversions, dicentrics) as well as a cell line monosomic for a D group chromosome and a pseudodiploid cell line monosomic for a no. 3 chromosome and trisomic for a C group chromosome. The various stem-lines were observed in cultures ranging from 41 to 144 days old and comprised varying percentages of the cell populations, ranging from less than 1% to almost 100%. DISCUSSION
By culturing blood lymphocytes and skin fibroblasts from a group of control women and OC users, we attempted to
Vol. 26, No.8
EFFECT OF ORAL CONTRACEPTIVES ON CHROMOSOMES
determine whether these synthetic hormones caused increased cytogenetic aberrations in human somatic cells other than lymphocytes. We chose to study fibroblasts because these cells are relatively easy to culture and are the only somatic cells other than lymphocytes that can be obtained with a minimum of trauma from human volunteers. We realized at the outset that there were some problems is using this cell type for estimates of aberrations induced in vivo. Because of the loss of cells bearing some types of aberrations and the possible evolution of stem-lines of other abnormal cells, it follows that the aberrations observed in fibroblasts cultured in vitro for a period of several weeks would not necessarily reflect the in vivo condition of this population of cells. Thus, in an effort to determine the effect of OC on chromosomes, we not only analyzed breakages in standard cultures exposed to FCS but also exposed replicate fibroblast cultures to autologous serum, homologous serum, and homologous serum from OC users. From the series of replicate cultures we hoped to learn whether the frequency of breakages or stable rearrangements was higher in the standard cultures from the OC users, whether fibroblasts from women who had taken OC would show increased breakages or abnormal growth when re-exposed to serum containing synthetic hormonal constituents, and whether serum from OC users contained substances that could induce breakages in fibroblasts from women who had not taken oral contraceptives. Results of cytogenetic analyses of these fibroblast cultures showed no difference in chromosome breakages in cells from OC users cultured in FCS, compared with control cells cultured in the same serum, and we could not detect any evidence of breakage induced by OC serum in either the control fibroblasts or fibroblasts from OC users. Likewise, we found no evidence
831
for increased frequency of cytogenetically abnormal stem-lines in fibroblasts from the women taking OC. In fact, we observed 5 aberrant clones of cells in 19 primary cultures derived from skin from the control women, and only 2 aberrant clones in 17 primary cultures from the OC users. We do not know whether the 7 abnormal cell lines observed in these cultures arose from cells that suffered injuries in vivo or whether the clones evolved as a result of an injury incurred during the period of culture, but the lack of observation of a substantial number of chromosomally aberrant stem-lines in the 17 sets of cultures from the OC users certainly suggests that fibroblasts from these women are no more predisposed to the evolution of aberrant clones than are fibroblasts from women who have not taken synthetic hormones. In the present series of experiments we did not observe an increase in chromosome breakages in lymphocyte cultures from five women taking OC, compared with breakages in cultures from five women who had never taken synthetic hormones. This findings is not unexpected since our larger survey study of aberrations in lymphocytes from pill users 6 had demonstrated considerable variability in breakages in consecutive cultures from the same individual, and among cultures from different persons. Likewise, we had previously found that average breakages in series of cultures from some OC users were not different from breakages in cultures from some control women. Because of the variability in breakages among cultures from the same individual, we obtained four separate sets of tissue samples from each woman for the evaluation of chromosome lesions. However, sl.nce we obtained blood from only five women in each group, it is possible that our sample size was not large enough to detect the minor increases in breakages that we had observed in the larger group of OC users.
832
LITTLEFIELD AND MAll..HES
SUMMARY
To determine whether exposure to synthetic hormones resulted in increased chromosome damage in cells other than lymphocytes, we evaluated series of lymphocyte and fibroblast cultures from five control women and five women taking oral contraceptives (OC). The results of this study showed: {1) no difference in chromosome breakages between lymphocytes and fibroblasts; (2) no differences in breakages in replicate fibroblast cultures exposed to fetal calf serum, autologous serum, homologous serum, or homologous serum from OC users; (3) no difference in breakages between lymphocyte and fibroblast cultures from OC users, compared with lymphocyte and fibroblast cultures from control women; and (4) no increase in the frequency of cytogenetically aberrant stem-lines in fibroblast cultures from women taking OC. These findings suggest that synthetic hormones do not cause increased chromosome breakages in fibroblasts from women taking OC. Acknowledgment. The authors gratefully acknowledge the technical assistance of Mr. Barry Lazar, Mr. Eugene Joiner, and Ms. Lydia Corrill, and express appreciation to Mr. F. L. Miller, who conducted the statistical analyses.
2.
3.
4. 5.
6.
7.
8.
9.
REFERENCES 1. Goh KO: Chromosomal breaks in women taking
birth control pills. In Research Report, 1967Medical Division, Oak Ridge Associated Uni-
10 ·
August 1975
versities. Oak Ridge, Tenn, US AEC Report ORAU-106, 1968, p 97 McQuarrie HG, Scott CD, Ellsworth HS, Harris JW, Stone RA: Cytogenetic studies on women using oral contraceptives and their progeny. Am J Obstet Gynecol108:659, 1970 Badr FM, Badr RS, lbrakin A, Shaaban HA: Effect of oral and injectable contraceptives on the human chromosomes. In Fourth International Congress of Human Genetics, Paris, 6-11 September 1971, Abstracts of Papers Presented (International Congress Series 233). Amsterdam, Excerpta Medica, 1971, p 20 Shapiro LR, Graves ZR, Hirschhorn K: Oral contraceptives and in vivo cytogenetic studies. Obstet Gynecol 39:190, 1972 de Gutierrez AC, Lisker R: Longitudinal study of the effects of oral contraceptives on human chromosomes. Ann Genet (Paris) 16:259, 1973 Littlefield LG, Lever WE, Miller FL, Goh KO: Chromosome-breakage studies in lymphocytes from normal women, pregnant women, and women taking oral contraceptives. Am J Obstet Gynecol 121:976, 1975 Littlefield LG, Mailhes J: Observations of de novo clones of cytogenetically aberrant cells in primary fibroblast cell strains from phenotypically normal women. Am J Hum Genet 27:190, 1975 Littlefield LG, Goh KO: Cytogenetic studies in control men and women. 1. Variations in aberration frequencies in 29,709 metaphases from 305 cultures obtained over a three-year period. Cytogenet Cell Genet 12:17, 1973 Mailhes JB, Lazar BS, Littlefield LG: A reliable procedure for the in vitro culture of human fibroblasts. In Research Report, 1971-Medical Division, Oak Ridge Associated Universities. Oak Ridge, Tenn, US AEC Report ORAU-122, 1973, p 177 Sasaki M, Norman A: Proliferation of human lymphocytes in culture. Nature 210:913, 1966
