Cytogenetics of methadone-managed and heroin-addicted pregnant women and their newborn infants ANTHONY
P.
MARIJA
J.
Chicago,
Illinois
AMAROSE,
PH.D.
NORUSIS,
PH.D.
Chromosomal damage was assessedprenatally and at delivery from 99 addicted Fegnant women (80 from a methadone maintenance flogram and 19 heroin addicts) and th&r 101 offspring at delivery. About IO per cent of the 27,907 cells scored showed chromosomal aberrations. Chromosome damage was random, affected all chromosomes, and was mainly of thp acentric fragment type. The per cent of hypodiploidy was significantly higher than the per cent of hyperdiplotiy. In the mothers, no sign$cant differences were found with respect to dosage and duration of methadone treatment and prars of heroin abuse. No signiJicant association was found between maternal variables arid infant chromosome damage. Infants with low Apgar scores (I to 6) had cells with sign$rantly higher levels of chromosome damage than infants with higher scores (7 to IO).
the potential genetic hazards of short- and long-term exposure to drugs of abuse has motivated efforts to identify those compounds associated with cytogenetic damage as well as to examine the possible effects on the organism of such damage. Some investigators have attributed chromosome alterations to drug abuse, ‘3 2 while others have not observed this relationship.3-6 A multiplicity of factors, such as CONCERN
chronic fatigue, poor health, and inadequate nutrition, usual concomitants of drug intoxication which may themselves be related to chromosome damage, compound the difficulties encountered in such investigations and may have contributed to the contradictory results reported.le6 The substitution of the synthetic narcotic drug methadone for heroin addiction, especially its prescrip tion during pregnancy, raises anew the cytogenetic questions long associated with illicit drug abuse. Residual chromosome damage has been reported in former heroin addicts before and during methadone treatment,’ but in vitro methadone cultures of newborn cord blood did not reveal an increase in chromosome damage.s The primary objective of this report is to consider the qualitative and quantitative cytogenetics of peripheral blood cultures from methadone-managed and heroin-addicted pregnant women and from the resulting offspring at birth.
ABOUT
From
thp Department
of Obstetrics
and Gynecology,
Medic&,
and Statistics, The University of Chicago, Pritzkr School of MediCk. Supported in part by Maternal Child Health Research Grant MC-R-1 70168 and the Otho Sprague Memorial Institutional Received R&sed Accepted
Grant.
for publication J+ July
June
9, 1975.
25, 1975. 31, 1975.
Reprint reqwsts: Dr. Anthony P. Amarose, Department Obstetrics and Gynecology, University of Chicago, 5841 Maryland Arw., Chicago, Illinois 60637.
of
635
636
March Am. J. Ohstet.
Amarose and Norusis
Table
I. Summary
chromosome
Type of sample Maternal
prenatal
Methadone Heroin Maternal
addicts
4
deliver?,
99
Heroin Infant
81 addicts
delivev
Methadone Heroin
*Gaps. tIncludes
80 76
Methadone
18 101 82
addicts
profiles Aberrant (%)
No.
19
15. 1976 Gynecol.
12.01t (0.63t) 12.10 (0.66) 10.25 (1.93) 12.56 (0.54) 12.95 (0.60) 10.83 (1.09) 12.48 (0.67) 12.52 (0.77) 12.32 (1.34)
cells
10.05 (0.60) 10.17 (0.62) 7.75 (2.14) 10.10 (0.51) 10.43 (0.58) 8.61 (1.07) 9.87 (0.54) 9.73 (0.61) 10.47 (1.21)
Aberrations1 aberrant cell 1.44 (0.11) 1.45 (0.12) 1.31 (0.04) 1.39 (0.04) 1.42 (0.04) 1.25 (0.04) 1.36 (0.03) 1.36 (0.03) 1.36 (0.05)
Aberratiolul I00 cells scored
Chromotid brrakr (%i
Chromatid kSiOlLS* (%I
14.11 (1.02) 14.31 (1.06) 10.25 (2.98) 14.34 (0.90) 15.05 (1.03) 11.11 (1.52) 14.10 (1.01) 14.03 (1.18) 14.37 (1.88)
6.25 (0.42) 6.33 (0.43) 4.75 (1.55) 6.49 (0.42) 6.74 (0.48) 5.39 (0.81) 6.38 (0.39) 6.34 (0.43) 6.58 (0.92)
2.33 (0.30) 2.31 (0.31) 2.75 (0.25) 3.00 (0.31) 3.02 (0.37) 2.72 (0.49) 3.20 (0.39) 3.38 (0.46) 2.42 (0.57)
lesions.
$Standard error.
Material and methods In response to an urgent need for information concerning the effects of narcotic addiction and methadone maintenance on pregnancy and the immediate postpartum period, the Departments of Obstetrics and Gynecology, Psychiatry, Pediatrics, and Medicine and the Illinois Drug Abuse Program instituted a multifaceted program of clinical and research investigations. The course of pregnancy in methadonemanaged and heroin-addicted women was studied for any possible effects of narcotics. Addicts were from the Chicago metropolitan area; 102 obtained methadone prenatally at different times during the pregnancy through the Illinois Drug Abuse Program (I.D.A.P.), and 21 women were heroin addicts who presented at the Chicago Lying-in Hospital only at delivery. Study patients were primarily Black (86.2 per cent), Caucasian (8.9%). Spanish (4.1 per cent), and other (0.8 per cent). The mean age was 25.1 years with a range of 16 to 44 years. Forty-two per cent of the women were between 21 and 25 years of age. There was an average of 1.9 living children per patient. The addiction histories, obtained by the cytogenetics staff, revealed heroin to be the primary drug of abuse for more than three years in 74.5 per cent of the population. Multiple-drug abuse was common for all subjects and included alcohol, marihuana, cocaine, and other hypnotics, sedatives, stimulants, and depressants. Illicit drug use during pregnancy for those on methadone maintenance was monitored by I.D.A.P.
The methadone dosage range for those in prenatal treatment was 0 to 80 mg. daily with an average daily dose of 32.7 mg. The times of entry into the I.D.A.P. were: prior to conception, 41.5 per cent; first trimester, 9.8 per cent; second trimester, 17.9 per cent; third trimester, 13.8 per cent; never entered (heroin addicts), 17.1 per cent. Cytogenetic samples were obtained from 80 methadone-managed women and 19 heroin-addicted women. A blood sample was drawn at the first clinic visit for those women who had prenatal visits. At delivery, samples of maternal and infant cord blood were obtained from all patients. All deliveries were attended by the cytogenetic staff, and all cultures were started immediately after the blood was obtained. In no instance was blood refrigerated for later culturing. Nearly all cultures had 100 cells scored for a total of 27,907 cells. Preparation of 72 hour blood cultures with the use of a whole blood micromethod, harvesting, scoring of the chromosome complements, and classification of chromosome aberrations were carried out as previously described.g All slides were coded so that the observer was unaware of any relationship to patients. To strengthen the impartiality, no observer continually scored slides from the spaced peripheral blood cultures of the same subject. The ploidy and number and type of aberrations in each cell scored were entered onto forms which permitted direct keypunching. Chromosome profiles for each sample, as well as statistical analyses, were
Volume Number
Cytogenetics of pregnant addicts and their offspring
124
637
6
Chromosome
aberrations (%) 4.43 (0.34) 4.50 (0.36) 3.00 (0.71) 4.32 (0.28) 4.44 (0.32) 3.78 (0.63) 4.12 (0.29) 4.10 (0.33) 4.21 (0.49)
Aneuploidy Hypodiploidy 4.00 (0.27) 4.09 (0.28) 2.25 (0.25) 4.78
(0.34) 5.00 (0.67) 5.84 (0.42) 6.17 (0.49) 4.42 (0.64)
(%) Hyperdiploidy 1.23 (0.14) 1.21 (0.14) 1.50 (0.87) 1.17 (0.12) 1.12 (0.14) 1.39 w;’ (0.18) 1.38 (0.21) 1.32 (0.25)
compiled with the Xerox Sigma V computer. Significance was based on the Student’s t test and Pearson correlation-coefficient statistics. RSSUitS
Summary chromosome profiles for all study cases subdivided by type of sample, as well as maternal drug of abuse, are presented in Table I. About 10 per cent of all cells scored had some type of aberration. This figure did not differ much among the maternal prenatal (10.05) maternal delivery (10.10) and newborn (9.87) samples. If cells with chromosome lesions (gaps) are also considered as damaged, the per cent of aberrant cells increases to 12.4 per cent, with all the types of samples undergoing a similar increase. The women not in the methadone program had a slightly lower per cent of aberrant cells than methadonemanaged women, but this difference was not statistically significant. Although examination of the per cent of aberrant cells does provide information as to the incidence of damaged cells, it does not quantify the extent of damage in an aberrant cell. For this purpose the ratio of the total number of aberrations to the total number of aberrant cells and to the total number of cells scored for each subject was computed. Although the number of aberrations per total cells scored does not alone permit isolation of extent of damage in an individual cell, it is a commonly used index which allows comparison of the present results to those reported in
the literature. On the average, there were about 14 aberrations for every 10 aberrant cells scored. This remained fairly constant for the different samples. However, the heroin-addicted women at delivery had a significantly lower rate of aberrant cells (1.25) than the methadone-managed group (1.42). There was also a statistically significant difference between these two groups at delivery when aberrations per total cells scored were considered. The heroin addicts had a rate of 11 per cent while those on methadone had a rate of 15 per cent. It is of interest to note that the chromosome damage of infants in the two groups did not differ as measured by any of the indices. When aneuploidy was determined, the per cent of hypodiploid cells was significantly higher than the per cent of hyderdiploid cells for all three sample types. The per cent of hypodiploidy was significantly higher in the newborn infants when compared to the two types of maternal samples. The extent to which infant chromosome damage can be predicted from maternal prenatal and/or delivery samples is also of particular interest. Fig. 1 is a scatterplot of the maternal delivery damage versus infant cord blood damage. Each of the points on the graph represents one or more mother-infant pairs. If there was a perfect association between maternal and infant damage, the points would cluster closely about a straight line or some other type of curve. As can be seen from Fig. 1, the points show a large amount of variability which cannot be explained by fitting any type of curve. However, even if the association between maternal and infant damage is not very strong, the question as to whether any degree of association exists can be considered. With the correlation coefficient, a measure of linear association, no relationship was found between the per cent of aberrant cells of the mother and newborn infant at delivery. When the per cent of aberrant cells including chromosome lesions was considered, there was a small (r = 0.26) but statistically significant (p < 0.01) association between maternal delivery and newborn damage. There was also a significant association between delivery maternal and newborn hypodiploidy (r = 0.25; p < 0.01) and diploidy (r = 0.25; p < 0.01). When the relationship between maternal prenatal and newborn samples was examined, no significant associations were found. The association between the two types of maternal samples was also weak, though statistically significant, for the per cent of aberrant cells without lesions (r = 0.26; p < 0.05), the per cent of aberrant cells with lesions (r = 0.19; p < 0.05), and the per cent of hyperdiploid cells (r = 0.19; p < 0.05).
838
March Am. J. Ohstet.
Amarose and Norusis
Table II. Chromosome Apgar scores
30 .
t
Apgar
.
z !i
l 16.
. . l
.
A k-z
l.
.
h
z 124-l
. .
A. . ..* A . .
. .
.
.
*A. .
4
.
:: . . .
.
.
. . . . .
8
Total
12
7-8
32
9-10
57
Total
89
.
0.
*
4-6
.
.
. .
:’
. OO
.
. l
.
NO.
.
‘0.. .:. 4.
!6-
.
.
score
One minute l-3
.
2
g iti
profile
.
I 4 24.
2
“=I00 r =o.o
: . .
.
’ .
l
.:
6
12
PER CENT MATERNAL
16
24
30
DELIVERY DAMAGE
Fig. 1. Relationship at delivery between per cent of maternal and infant cells with chromosome damage. Symbols representing the number of mother-infant pairs are: l = 1: A = 2.
Also considered was whether poor infant outcome-whether it be depressed Apgar scores, intrauterine growth retardation, or presence of withdrawal symptoms severe enough to necessitate chemotherapeutic intervention-was also associated with an increase in chromosome damage. Only Apgar scores appeared to have some type of association with chromosome damage (Table II). Infants with low Apgar scores had more chromosome damage than infants with high Apgar scores. There was a significant difference in the number of aberrations per aberrant cell for infants with one- and five-minute Apgar scores of 6 or less when compared to infants with scores of 7 to 10. Infants were also subdivided by a variety of maternal characteristics which might affect infant chromosome damage: duration of time heroin was used as a primary drug, type of primary drug at conception and delivery, and duration and dosage of methadone treatment. Infants born to mothers addicted to heroin appeared not to differ significantly from infants born to methadone-managed mothers. No consistent difference with respect to duration of reported maternal heroin usage or methadone dosages could be found. Both the maternal prenatal and delivery samples were subdivided by the same characteristics as the infant samples. Again, no clear-cut differences emerged with respect to dosage, duration of methadone treatment, and years of heroin use, although, as shown in Table 1, the women who did not enter the methadone program had fewer aberrant cells. When particular types of chromatid and chromosome aberrations were scored, the prevalent type in all cells was the chromatid break and lesion. Chromosome
Five
of infants
Aberrant cells C%)
13, 1976 Gynecol.
subdivided
h\
Aberrations1 aberrant cdl
20.0* (5.7lt) 14.5 (2.70) 16.3 (2.58) 12.6 (1.30) 11.6 (0.75) 12.0 (0.67)
17.5 (4.44) 11.2 (2.53) 13.3 (2.30) 9.6 (0.95) 9.3 (0.61) 9.4
(0.52)
1.83 (0.15) 1.43 (0.13) 1.57 (0.1 I) 1.39 (0.05) 1.30 (0.03) 1.33 (0.03)
36.0 (4 11.0 (2.97) 15.2 (4.82) 16.7 (2.39) 12.0 (0.67) 12.3 (0.66)
29.0 (-) 10.4 (2.87) 13.5 (3.89) 13.2 (2.14) 9.4 (0.53) 9.6 (0.52)
2.28 (-) 1.60 (0.06) I .72 (0.12) 1.28 (0.07) I .34 (0.03) I .34 (0.03)
minute l-3
1
4-6
5
Total
6
7-8
6
9-10
89
Total
95
*Includes tstandard
lesions. error.
damage (involving both sister chromatids) was predominantly that of the isochromatid break (ace). There were no standard abnormal karyotypes, clones of abnormal cells, or marker chromosomes. Chromosome damage was random and involved all chromosones.
Comment In view of the difficulties encountered with the identification of in vivo chromosome-altering agents, progression to yet another generation, the newborn infant, compounds the already complex problem. When the infant is of drug-addicted parentage, the number of possible variables which might influence chromosome damage increases greatly, especially since there have been no similar studies in nonaddict mother/infant pairs. The degree of maternal prenatal and delivery chromosome damage explained very little of the variation observed in the newborn infant, suggesting several possible mechanisms. For example, the factors which cause chromosome damage ma) affect the mother and infant differently. Although at conception the levels of maternal and fetal damage may have been comparable, continued exposure of both to varied stresses may have resulted in differing chromosome profiles at delivery. The weak association between maternal samples prenatallv and at delivery
Volume Number
124 6
indicates that maternal chromosome damage did not remain stable throughout the prenatal period but fluctuated over relatively short time periods. Whether a similar situation would exist in drug-free pregnant women remains unknown. The failure to establish an association between the level of the various drugrelated variables and the degree of chromosome damage may suggest there is none. Equally likely is the possibility that numerous complex interactions of both measured and unmeasured variables obscured the examined relationships. Perhaps under more carefully monitored life-style conditions different results would have been obtained. It must also be emphasized that since nonaddict infant cord blood and maternal measurements are unavailable it cannot be established whether our reported values are significantly different from those that would be observed in matched control subjects. The increased incidence of chromosomal damage in infants with low Apgar scores suggests that elevated chromosome damage may be concomitant with differing types of neonatal difficulties associated with depressed Apgar scores. The exact role of the observed chromosomal damage in such situatians is unknown. It is conceivable that chromosomal damage may have led to the neonatal difficulties or that the difficulties could have contributed to the increased damage. Although no discernible differences in infant chromosome profiles were seen between infants requiring treatment for withdrawal and those who did not, the very small number of infants judged to have no withdrawal and delayed onset of withdrawal in many, as well as the possibility of previous in utero withdrawal, makes any definitive conclusions tenuous. The reported frequency of aberrant cells in drug users and control subjects has ranged from 1 to 20 per cent.** I49 I5 In this study the incidence of the particular types of chromosome damage was lower in all but one category (isochromosomes) when compared to the evaluations of 2,237 cells from former illicit drug users.6 Although the over-all per cent distribution of isochromosomes was slightly higher, there were only two ring chromosomes observed in the 27,907 cells scored. The biological significance of chromatid gaps is not known, but continued notation of these appears required in view of evidence of association of specific diseases with specific and consistent achromatic gaps. Three patients with congenital erythroid hypoplasia
Cytcgenetics of pregnant addicts and their offspring
639
(Blackfan-Diamond syndrome) and an abnormality of chromosome no. 1 have been reported.10-‘3 One patient had an achromatic gap in the same region of one arm of Chromosome l.*“, ‘I The patients of Gray and associates” and Heyn and colleagues13 also had an abnormality in the same chromosome. These three studies relate the chromatid gap to a specific disease. In this study we originally included chromatid lesions in Fig. 1, and the relationship of maternal delivery/infant delivery chromosome damage was statistically significant. When the chromatid gaps were removed, the results did not show statistical significance. One of the principal concerns in drug abuse is the possible production of irreversible chromosome damage which could be deleterious. Although chromosomal changes in general are not pathognomonic of narcotics abuse, the increased incidence of chromosomal abnormalities in the lymphocytes from narcotics abusers when compared to non-drug users strengthens the possibility of a relationship. The life-styles of the 99 subjects did not demonstrate a single instance of aseptic administration of the drugs, and there is no way we can prove the purity of the preparations. Compounding the drug addiction was the presence of poor nutrition, lack of rest, stress, increased incidence of tuberculosis, hepatitis, and syphilis, and a lack of early and consistent prenatal care. For these reasons as well as simultaneous poly-drug use, it is impossible to isolate either methadone or heroin as the causative agent. Conceptually, it is possible that any of these factors could have damaged the detoxifying potential of the liver to such an extent that the small lymphocytes became “fragile,” “infected,” or “primed” so that in vitro stimulation into mitosis either elicited existing residual chromosomal damage or engendered chromosome damage in these weakened cells within the in vitro milieu. In summary, the biological implications of chromosome damage cannot be completely understood until it can be ascertained how the human subject reacts to chromosome damage when there is no evidence of clinical or pathologic syndromes. We are indebted to the faculty, residents, and staff of the Department of Obstetrics and Gynecology for their understanding and cooperation during a difficult project and to Cecilia Turriff, Mitchell Kraynak, and Thomas Diamante for technical assistance.
REFERENCES
1. Cohen, M. M., Marinello, M. J., and Beck, N.: Chromosomal damage in human leukocytes induced by lysergic acid diethylamide, Science 155: 1417, 1967.
2. Irwin, S., and Egozcue, J.: Chromosomal abnormalities in leukocytes from LSD-25 users, Science 157: 313, 1967. 3. Hungerford, D. A., Taylor, K. M., Shagass, C., LaBadie,
640
4.
5.
6.
7.
8.
9.
Amarose and Norusis
G. U., Balaban, G. B., and Paton, G. R.: Cytogenetic effects of LSD 25 therapy in man, J. A. M. A. 206: 2287, 1968. Tjio, J. H., Pahnke, W. M., and Kurland, A. A.: LSD and chromosomes: A controlled experiment, J. A. M. A. 210: 849, 1969. Dishotsky, N. I., Loughman, W. D., Mogar, R. E., and Lipscomb, W. R.: LSD and genetic damage, Science 172: 431, 1971. Amarose, A. P., and Schuster, C. R.: Chromosome analyses of bone marrow and peripheral blood in subjects with a history of illicit drug use, Arch. Gen. Psychiatry 25: 181, 1971. Falek, A., and McFadden, I. J.: Cytogenetic follow-up of patients in a methadone mamtenan& program: A pilot studv, in DuPont, R. L.. and Freeman. R. S.. editors: 1973 Proceedings of the National Association for the Prevention of Addiction to narcotics, New York, 1973, pp. 695-705. Falek, A., Jordan, R. B., King, B. J., Arnold, P. J., and Skelton, W. D.: Human chromosomes and opiates, Arch. Gen. Psychiatry 27: 511, 1972. Amarose, A. P., Plotz, E. J., and Stein, A. A.: Residual chromosomal aberrations in female cancer patients
March Am. J. Obstet.
15, 1976 Gynecol.
following irradiation therapy, Exp. Mol. Pathol. 7: 58, 1967. 10. Amarose, A. P., Tartaglia, A. P., and Propp, S.: Cytogenetic findings in Blackfan-Diamond syndrome, Lancet 2: 1020, 1965. 11. Tartaglia, A. P., Propp, S., Amarose, A. P., Propp, R. P., and Hall, C. A.: Chromosome abnormality and hypocalcemia in congenital erythroid hypoplasia (Bl&kfanDiamond svndrome), Am. I. Med. 41: 990, 1966. 12. Gray, J. E.,‘Syrett, J.‘E., Ritihie, K. M., and Elliott, W. D.: An interstitial transiocation: Chromosome no. lp to 4q, Lancet 2: 92, 1972. 13. Heyn, R., Kurczynski, E., and Schmickel, R.: The association of Blackfan-Diamond syndrome, physical abnormalities, and an abnormality of chromosome 1, J. Pediatr. 85: 531, 1974. 14. Littlefield, L. G., and Goh, K. 0.: Cytogenetic studies in control men and women. I. Variations in aberration frequencies in 29,709 metaphases from 305 cultures obtained over a three-year period, Cytogenet. Cell Genet. 12: 17, 1973. 15. Lubs. H. A., and Samuelson, J.: Chromosome abnormalities in lymphocytes from normal human subjects, Cytogenetics 6: 402, 1967.
P.
MARIJA
J.
Chicago,
Illinois
AMAROSE,
PH.D.
NORUSIS,
PH.D.
Chromosomal damage was assessedprenatally and at delivery from 99 addicted Fegnant women (80 from a methadone maintenance flogram and 19 heroin addicts) and th&r 101 offspring at delivery. About IO per cent of the 27,907 cells scored showed chromosomal aberrations. Chromosome damage was random, affected all chromosomes, and was mainly of thp acentric fragment type. The per cent of hypodiploidy was significantly higher than the per cent of hyperdiplotiy. In the mothers, no sign$cant differences were found with respect to dosage and duration of methadone treatment and prars of heroin abuse. No signiJicant association was found between maternal variables arid infant chromosome damage. Infants with low Apgar scores (I to 6) had cells with sign$rantly higher levels of chromosome damage than infants with higher scores (7 to IO).
the potential genetic hazards of short- and long-term exposure to drugs of abuse has motivated efforts to identify those compounds associated with cytogenetic damage as well as to examine the possible effects on the organism of such damage. Some investigators have attributed chromosome alterations to drug abuse, ‘3 2 while others have not observed this relationship.3-6 A multiplicity of factors, such as CONCERN
chronic fatigue, poor health, and inadequate nutrition, usual concomitants of drug intoxication which may themselves be related to chromosome damage, compound the difficulties encountered in such investigations and may have contributed to the contradictory results reported.le6 The substitution of the synthetic narcotic drug methadone for heroin addiction, especially its prescrip tion during pregnancy, raises anew the cytogenetic questions long associated with illicit drug abuse. Residual chromosome damage has been reported in former heroin addicts before and during methadone treatment,’ but in vitro methadone cultures of newborn cord blood did not reveal an increase in chromosome damage.s The primary objective of this report is to consider the qualitative and quantitative cytogenetics of peripheral blood cultures from methadone-managed and heroin-addicted pregnant women and from the resulting offspring at birth.
ABOUT
From
thp Department
of Obstetrics
and Gynecology,
Medic&,
and Statistics, The University of Chicago, Pritzkr School of MediCk. Supported in part by Maternal Child Health Research Grant MC-R-1 70168 and the Otho Sprague Memorial Institutional Received R&sed Accepted
Grant.
for publication J+ July
June
9, 1975.
25, 1975. 31, 1975.
Reprint reqwsts: Dr. Anthony P. Amarose, Department Obstetrics and Gynecology, University of Chicago, 5841 Maryland Arw., Chicago, Illinois 60637.
of
635
636
March Am. J. Ohstet.
Amarose and Norusis
Table
I. Summary
chromosome
Type of sample Maternal
prenatal
Methadone Heroin Maternal
addicts
4
deliver?,
99
Heroin Infant
81 addicts
delivev
Methadone Heroin
*Gaps. tIncludes
80 76
Methadone
18 101 82
addicts
profiles Aberrant (%)
No.
19
15. 1976 Gynecol.
12.01t (0.63t) 12.10 (0.66) 10.25 (1.93) 12.56 (0.54) 12.95 (0.60) 10.83 (1.09) 12.48 (0.67) 12.52 (0.77) 12.32 (1.34)
cells
10.05 (0.60) 10.17 (0.62) 7.75 (2.14) 10.10 (0.51) 10.43 (0.58) 8.61 (1.07) 9.87 (0.54) 9.73 (0.61) 10.47 (1.21)
Aberrations1 aberrant cell 1.44 (0.11) 1.45 (0.12) 1.31 (0.04) 1.39 (0.04) 1.42 (0.04) 1.25 (0.04) 1.36 (0.03) 1.36 (0.03) 1.36 (0.05)
Aberratiolul I00 cells scored
Chromotid brrakr (%i
Chromatid kSiOlLS* (%I
14.11 (1.02) 14.31 (1.06) 10.25 (2.98) 14.34 (0.90) 15.05 (1.03) 11.11 (1.52) 14.10 (1.01) 14.03 (1.18) 14.37 (1.88)
6.25 (0.42) 6.33 (0.43) 4.75 (1.55) 6.49 (0.42) 6.74 (0.48) 5.39 (0.81) 6.38 (0.39) 6.34 (0.43) 6.58 (0.92)
2.33 (0.30) 2.31 (0.31) 2.75 (0.25) 3.00 (0.31) 3.02 (0.37) 2.72 (0.49) 3.20 (0.39) 3.38 (0.46) 2.42 (0.57)
lesions.
$Standard error.
Material and methods In response to an urgent need for information concerning the effects of narcotic addiction and methadone maintenance on pregnancy and the immediate postpartum period, the Departments of Obstetrics and Gynecology, Psychiatry, Pediatrics, and Medicine and the Illinois Drug Abuse Program instituted a multifaceted program of clinical and research investigations. The course of pregnancy in methadonemanaged and heroin-addicted women was studied for any possible effects of narcotics. Addicts were from the Chicago metropolitan area; 102 obtained methadone prenatally at different times during the pregnancy through the Illinois Drug Abuse Program (I.D.A.P.), and 21 women were heroin addicts who presented at the Chicago Lying-in Hospital only at delivery. Study patients were primarily Black (86.2 per cent), Caucasian (8.9%). Spanish (4.1 per cent), and other (0.8 per cent). The mean age was 25.1 years with a range of 16 to 44 years. Forty-two per cent of the women were between 21 and 25 years of age. There was an average of 1.9 living children per patient. The addiction histories, obtained by the cytogenetics staff, revealed heroin to be the primary drug of abuse for more than three years in 74.5 per cent of the population. Multiple-drug abuse was common for all subjects and included alcohol, marihuana, cocaine, and other hypnotics, sedatives, stimulants, and depressants. Illicit drug use during pregnancy for those on methadone maintenance was monitored by I.D.A.P.
The methadone dosage range for those in prenatal treatment was 0 to 80 mg. daily with an average daily dose of 32.7 mg. The times of entry into the I.D.A.P. were: prior to conception, 41.5 per cent; first trimester, 9.8 per cent; second trimester, 17.9 per cent; third trimester, 13.8 per cent; never entered (heroin addicts), 17.1 per cent. Cytogenetic samples were obtained from 80 methadone-managed women and 19 heroin-addicted women. A blood sample was drawn at the first clinic visit for those women who had prenatal visits. At delivery, samples of maternal and infant cord blood were obtained from all patients. All deliveries were attended by the cytogenetic staff, and all cultures were started immediately after the blood was obtained. In no instance was blood refrigerated for later culturing. Nearly all cultures had 100 cells scored for a total of 27,907 cells. Preparation of 72 hour blood cultures with the use of a whole blood micromethod, harvesting, scoring of the chromosome complements, and classification of chromosome aberrations were carried out as previously described.g All slides were coded so that the observer was unaware of any relationship to patients. To strengthen the impartiality, no observer continually scored slides from the spaced peripheral blood cultures of the same subject. The ploidy and number and type of aberrations in each cell scored were entered onto forms which permitted direct keypunching. Chromosome profiles for each sample, as well as statistical analyses, were
Volume Number
Cytogenetics of pregnant addicts and their offspring
124
637
6
Chromosome
aberrations (%) 4.43 (0.34) 4.50 (0.36) 3.00 (0.71) 4.32 (0.28) 4.44 (0.32) 3.78 (0.63) 4.12 (0.29) 4.10 (0.33) 4.21 (0.49)
Aneuploidy Hypodiploidy 4.00 (0.27) 4.09 (0.28) 2.25 (0.25) 4.78
(0.34) 5.00 (0.67) 5.84 (0.42) 6.17 (0.49) 4.42 (0.64)
(%) Hyperdiploidy 1.23 (0.14) 1.21 (0.14) 1.50 (0.87) 1.17 (0.12) 1.12 (0.14) 1.39 w;’ (0.18) 1.38 (0.21) 1.32 (0.25)
compiled with the Xerox Sigma V computer. Significance was based on the Student’s t test and Pearson correlation-coefficient statistics. RSSUitS
Summary chromosome profiles for all study cases subdivided by type of sample, as well as maternal drug of abuse, are presented in Table I. About 10 per cent of all cells scored had some type of aberration. This figure did not differ much among the maternal prenatal (10.05) maternal delivery (10.10) and newborn (9.87) samples. If cells with chromosome lesions (gaps) are also considered as damaged, the per cent of aberrant cells increases to 12.4 per cent, with all the types of samples undergoing a similar increase. The women not in the methadone program had a slightly lower per cent of aberrant cells than methadonemanaged women, but this difference was not statistically significant. Although examination of the per cent of aberrant cells does provide information as to the incidence of damaged cells, it does not quantify the extent of damage in an aberrant cell. For this purpose the ratio of the total number of aberrations to the total number of aberrant cells and to the total number of cells scored for each subject was computed. Although the number of aberrations per total cells scored does not alone permit isolation of extent of damage in an individual cell, it is a commonly used index which allows comparison of the present results to those reported in
the literature. On the average, there were about 14 aberrations for every 10 aberrant cells scored. This remained fairly constant for the different samples. However, the heroin-addicted women at delivery had a significantly lower rate of aberrant cells (1.25) than the methadone-managed group (1.42). There was also a statistically significant difference between these two groups at delivery when aberrations per total cells scored were considered. The heroin addicts had a rate of 11 per cent while those on methadone had a rate of 15 per cent. It is of interest to note that the chromosome damage of infants in the two groups did not differ as measured by any of the indices. When aneuploidy was determined, the per cent of hypodiploid cells was significantly higher than the per cent of hyderdiploid cells for all three sample types. The per cent of hypodiploidy was significantly higher in the newborn infants when compared to the two types of maternal samples. The extent to which infant chromosome damage can be predicted from maternal prenatal and/or delivery samples is also of particular interest. Fig. 1 is a scatterplot of the maternal delivery damage versus infant cord blood damage. Each of the points on the graph represents one or more mother-infant pairs. If there was a perfect association between maternal and infant damage, the points would cluster closely about a straight line or some other type of curve. As can be seen from Fig. 1, the points show a large amount of variability which cannot be explained by fitting any type of curve. However, even if the association between maternal and infant damage is not very strong, the question as to whether any degree of association exists can be considered. With the correlation coefficient, a measure of linear association, no relationship was found between the per cent of aberrant cells of the mother and newborn infant at delivery. When the per cent of aberrant cells including chromosome lesions was considered, there was a small (r = 0.26) but statistically significant (p < 0.01) association between maternal delivery and newborn damage. There was also a significant association between delivery maternal and newborn hypodiploidy (r = 0.25; p < 0.01) and diploidy (r = 0.25; p < 0.01). When the relationship between maternal prenatal and newborn samples was examined, no significant associations were found. The association between the two types of maternal samples was also weak, though statistically significant, for the per cent of aberrant cells without lesions (r = 0.26; p < 0.05), the per cent of aberrant cells with lesions (r = 0.19; p < 0.05), and the per cent of hyperdiploid cells (r = 0.19; p < 0.05).
838
March Am. J. Ohstet.
Amarose and Norusis
Table II. Chromosome Apgar scores
30 .
t
Apgar
.
z !i
l 16.
. . l
.
A k-z
l.
.
h
z 124-l
. .
A. . ..* A . .
. .
.
.
*A. .
4
.
:: . . .
.
.
. . . . .
8
Total
12
7-8
32
9-10
57
Total
89
.
0.
*
4-6
.
.
. .
:’
. OO
.
. l
.
NO.
.
‘0.. .:. 4.
!6-
.
.
score
One minute l-3
.
2
g iti
profile
.
I 4 24.
2
“=I00 r =o.o
: . .
.
’ .
l
.:
6
12
PER CENT MATERNAL
16
24
30
DELIVERY DAMAGE
Fig. 1. Relationship at delivery between per cent of maternal and infant cells with chromosome damage. Symbols representing the number of mother-infant pairs are: l = 1: A = 2.
Also considered was whether poor infant outcome-whether it be depressed Apgar scores, intrauterine growth retardation, or presence of withdrawal symptoms severe enough to necessitate chemotherapeutic intervention-was also associated with an increase in chromosome damage. Only Apgar scores appeared to have some type of association with chromosome damage (Table II). Infants with low Apgar scores had more chromosome damage than infants with high Apgar scores. There was a significant difference in the number of aberrations per aberrant cell for infants with one- and five-minute Apgar scores of 6 or less when compared to infants with scores of 7 to 10. Infants were also subdivided by a variety of maternal characteristics which might affect infant chromosome damage: duration of time heroin was used as a primary drug, type of primary drug at conception and delivery, and duration and dosage of methadone treatment. Infants born to mothers addicted to heroin appeared not to differ significantly from infants born to methadone-managed mothers. No consistent difference with respect to duration of reported maternal heroin usage or methadone dosages could be found. Both the maternal prenatal and delivery samples were subdivided by the same characteristics as the infant samples. Again, no clear-cut differences emerged with respect to dosage, duration of methadone treatment, and years of heroin use, although, as shown in Table 1, the women who did not enter the methadone program had fewer aberrant cells. When particular types of chromatid and chromosome aberrations were scored, the prevalent type in all cells was the chromatid break and lesion. Chromosome
Five
of infants
Aberrant cells C%)
13, 1976 Gynecol.
subdivided
h\
Aberrations1 aberrant cdl
20.0* (5.7lt) 14.5 (2.70) 16.3 (2.58) 12.6 (1.30) 11.6 (0.75) 12.0 (0.67)
17.5 (4.44) 11.2 (2.53) 13.3 (2.30) 9.6 (0.95) 9.3 (0.61) 9.4
(0.52)
1.83 (0.15) 1.43 (0.13) 1.57 (0.1 I) 1.39 (0.05) 1.30 (0.03) 1.33 (0.03)
36.0 (4 11.0 (2.97) 15.2 (4.82) 16.7 (2.39) 12.0 (0.67) 12.3 (0.66)
29.0 (-) 10.4 (2.87) 13.5 (3.89) 13.2 (2.14) 9.4 (0.53) 9.6 (0.52)
2.28 (-) 1.60 (0.06) I .72 (0.12) 1.28 (0.07) I .34 (0.03) I .34 (0.03)
minute l-3
1
4-6
5
Total
6
7-8
6
9-10
89
Total
95
*Includes tstandard
lesions. error.
damage (involving both sister chromatids) was predominantly that of the isochromatid break (ace). There were no standard abnormal karyotypes, clones of abnormal cells, or marker chromosomes. Chromosome damage was random and involved all chromosones.
Comment In view of the difficulties encountered with the identification of in vivo chromosome-altering agents, progression to yet another generation, the newborn infant, compounds the already complex problem. When the infant is of drug-addicted parentage, the number of possible variables which might influence chromosome damage increases greatly, especially since there have been no similar studies in nonaddict mother/infant pairs. The degree of maternal prenatal and delivery chromosome damage explained very little of the variation observed in the newborn infant, suggesting several possible mechanisms. For example, the factors which cause chromosome damage ma) affect the mother and infant differently. Although at conception the levels of maternal and fetal damage may have been comparable, continued exposure of both to varied stresses may have resulted in differing chromosome profiles at delivery. The weak association between maternal samples prenatallv and at delivery
Volume Number
124 6
indicates that maternal chromosome damage did not remain stable throughout the prenatal period but fluctuated over relatively short time periods. Whether a similar situation would exist in drug-free pregnant women remains unknown. The failure to establish an association between the level of the various drugrelated variables and the degree of chromosome damage may suggest there is none. Equally likely is the possibility that numerous complex interactions of both measured and unmeasured variables obscured the examined relationships. Perhaps under more carefully monitored life-style conditions different results would have been obtained. It must also be emphasized that since nonaddict infant cord blood and maternal measurements are unavailable it cannot be established whether our reported values are significantly different from those that would be observed in matched control subjects. The increased incidence of chromosomal damage in infants with low Apgar scores suggests that elevated chromosome damage may be concomitant with differing types of neonatal difficulties associated with depressed Apgar scores. The exact role of the observed chromosomal damage in such situatians is unknown. It is conceivable that chromosomal damage may have led to the neonatal difficulties or that the difficulties could have contributed to the increased damage. Although no discernible differences in infant chromosome profiles were seen between infants requiring treatment for withdrawal and those who did not, the very small number of infants judged to have no withdrawal and delayed onset of withdrawal in many, as well as the possibility of previous in utero withdrawal, makes any definitive conclusions tenuous. The reported frequency of aberrant cells in drug users and control subjects has ranged from 1 to 20 per cent.** I49 I5 In this study the incidence of the particular types of chromosome damage was lower in all but one category (isochromosomes) when compared to the evaluations of 2,237 cells from former illicit drug users.6 Although the over-all per cent distribution of isochromosomes was slightly higher, there were only two ring chromosomes observed in the 27,907 cells scored. The biological significance of chromatid gaps is not known, but continued notation of these appears required in view of evidence of association of specific diseases with specific and consistent achromatic gaps. Three patients with congenital erythroid hypoplasia
Cytcgenetics of pregnant addicts and their offspring
639
(Blackfan-Diamond syndrome) and an abnormality of chromosome no. 1 have been reported.10-‘3 One patient had an achromatic gap in the same region of one arm of Chromosome l.*“, ‘I The patients of Gray and associates” and Heyn and colleagues13 also had an abnormality in the same chromosome. These three studies relate the chromatid gap to a specific disease. In this study we originally included chromatid lesions in Fig. 1, and the relationship of maternal delivery/infant delivery chromosome damage was statistically significant. When the chromatid gaps were removed, the results did not show statistical significance. One of the principal concerns in drug abuse is the possible production of irreversible chromosome damage which could be deleterious. Although chromosomal changes in general are not pathognomonic of narcotics abuse, the increased incidence of chromosomal abnormalities in the lymphocytes from narcotics abusers when compared to non-drug users strengthens the possibility of a relationship. The life-styles of the 99 subjects did not demonstrate a single instance of aseptic administration of the drugs, and there is no way we can prove the purity of the preparations. Compounding the drug addiction was the presence of poor nutrition, lack of rest, stress, increased incidence of tuberculosis, hepatitis, and syphilis, and a lack of early and consistent prenatal care. For these reasons as well as simultaneous poly-drug use, it is impossible to isolate either methadone or heroin as the causative agent. Conceptually, it is possible that any of these factors could have damaged the detoxifying potential of the liver to such an extent that the small lymphocytes became “fragile,” “infected,” or “primed” so that in vitro stimulation into mitosis either elicited existing residual chromosomal damage or engendered chromosome damage in these weakened cells within the in vitro milieu. In summary, the biological implications of chromosome damage cannot be completely understood until it can be ascertained how the human subject reacts to chromosome damage when there is no evidence of clinical or pathologic syndromes. We are indebted to the faculty, residents, and staff of the Department of Obstetrics and Gynecology for their understanding and cooperation during a difficult project and to Cecilia Turriff, Mitchell Kraynak, and Thomas Diamante for technical assistance.
REFERENCES
1. Cohen, M. M., Marinello, M. J., and Beck, N.: Chromosomal damage in human leukocytes induced by lysergic acid diethylamide, Science 155: 1417, 1967.
2. Irwin, S., and Egozcue, J.: Chromosomal abnormalities in leukocytes from LSD-25 users, Science 157: 313, 1967. 3. Hungerford, D. A., Taylor, K. M., Shagass, C., LaBadie,
640
4.
5.
6.
7.
8.
9.
Amarose and Norusis
G. U., Balaban, G. B., and Paton, G. R.: Cytogenetic effects of LSD 25 therapy in man, J. A. M. A. 206: 2287, 1968. Tjio, J. H., Pahnke, W. M., and Kurland, A. A.: LSD and chromosomes: A controlled experiment, J. A. M. A. 210: 849, 1969. Dishotsky, N. I., Loughman, W. D., Mogar, R. E., and Lipscomb, W. R.: LSD and genetic damage, Science 172: 431, 1971. Amarose, A. P., and Schuster, C. R.: Chromosome analyses of bone marrow and peripheral blood in subjects with a history of illicit drug use, Arch. Gen. Psychiatry 25: 181, 1971. Falek, A., and McFadden, I. J.: Cytogenetic follow-up of patients in a methadone mamtenan& program: A pilot studv, in DuPont, R. L.. and Freeman. R. S.. editors: 1973 Proceedings of the National Association for the Prevention of Addiction to narcotics, New York, 1973, pp. 695-705. Falek, A., Jordan, R. B., King, B. J., Arnold, P. J., and Skelton, W. D.: Human chromosomes and opiates, Arch. Gen. Psychiatry 27: 511, 1972. Amarose, A. P., Plotz, E. J., and Stein, A. A.: Residual chromosomal aberrations in female cancer patients
March Am. J. Obstet.
15, 1976 Gynecol.
following irradiation therapy, Exp. Mol. Pathol. 7: 58, 1967. 10. Amarose, A. P., Tartaglia, A. P., and Propp, S.: Cytogenetic findings in Blackfan-Diamond syndrome, Lancet 2: 1020, 1965. 11. Tartaglia, A. P., Propp, S., Amarose, A. P., Propp, R. P., and Hall, C. A.: Chromosome abnormality and hypocalcemia in congenital erythroid hypoplasia (Bl&kfanDiamond svndrome), Am. I. Med. 41: 990, 1966. 12. Gray, J. E.,‘Syrett, J.‘E., Ritihie, K. M., and Elliott, W. D.: An interstitial transiocation: Chromosome no. lp to 4q, Lancet 2: 92, 1972. 13. Heyn, R., Kurczynski, E., and Schmickel, R.: The association of Blackfan-Diamond syndrome, physical abnormalities, and an abnormality of chromosome 1, J. Pediatr. 85: 531, 1974. 14. Littlefield, L. G., and Goh, K. 0.: Cytogenetic studies in control men and women. I. Variations in aberration frequencies in 29,709 metaphases from 305 cultures obtained over a three-year period, Cytogenet. Cell Genet. 12: 17, 1973. 15. Lubs. H. A., and Samuelson, J.: Chromosome abnormalities in lymphocytes from normal human subjects, Cytogenetics 6: 402, 1967.
