Mutation Research 586 (2005) 68–75
Micronucleus frequency in human umbilical cord lymphocytes M. Levario-Carrillo a , M. Sordo b , F. Rocha c , C. Gonz´alez-Horta d , D. Amato e , P. Ostrosky-Wegman b,∗ a
Unidad de Investigaci´on M´edica en Epidemiolog´ıa Cl´ınica, Instituto Mexicano del Seguro Social (IMSS), Chihuahua, M´exico b Department of Genomic Medicine and Environmental Toxicology, Instituto de Investigaciones Biom´ edicas, UNAM, P.O. Box 70228, Ciudad Universitaria, M´exico 04510, D.F., M´exico c Instituto Nacional de Perinatolog´ıa, SSa, M´ exico, D.F., M´exico d Facultad de Ciencias Qu´ımicas, Universidad Aut´ onoma de Chihuahua, Chihuahua, M´exico e Centro M´ edico Siglo XXI, IMSS, M´exico, D.F., M´exico Received 15 May 2004; received in revised form 24 May 2005; accepted 27 May 2005 Available online 7 July 2005
Abstract The human fetus is exposed to a variety of environmental agents and drugs which cross the placenta and can induce DNA damage. Micronucleus (MN) determination is a suitable and sensitive method for measuring DNA damage and since umbilical cord blood is obtained without any risk for the newborn, we measured the frequency of MN in cells from cord blood in four groups of healthy newborns (NB): 35 NB whose mothers lived in two urban cities (groups I and II); 16 NB from an agricultural area (group III); and 15 NB of mothers with high-risk pregnancy (group IV). MN were also evaluated in the mothers of NB from group I (n = 17) and group III (n = 14). Acetylcholinesterase (AChE) concentration was measured in groups I and III. The average frequency of binucleated cells with MN was 3.7 ± 1.4 in 1000 cells in mothers and 1 ± 0.9 in 1000 cells in NB from urban areas; and 4.5 ± 2.4 in1000 cells in mothers and 2 ± 1.5 in 1000 cells in NB from the agricultural area. The correlation between the frequency of MN in mothers and NB was significant (r = 0.61, p < 0.01). AChE levels of samples obtained both from group III mothers and from newborns were similar to those of group I. The Wilcoxon’s rank-sum test was applied to measure differences in MN frequency; NB of group I were used as control group. A significant (p < 0.01) higher frequency of MN (4 ± 2) was found only in lymphocytes from NB from high-risk pregnancies. Data indicate that MN evaluation in umbilical cord samples might be useful in the identification of transplacental mutagens. © 2005 Elsevier B.V. All rights reserved. Keywords: Micronuclei; Umbilical cord blood; Pesticides; High-risk pregnancy
1. Introduction ∗ Corresponding author. Tel.: +52 55 5622 3846; fax: +52 55 5622 3365. E-mail address:
[email protected] (P. Ostrosky-Wegman).
1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.05.006
The human fetus is occasionally exposed to environmental agents or to drugs administered to the mother for therapeutic or prophylactic purposes, which can cross
M. Levario-Carrillo et al. / Mutation Research 586 (2005) 68–75
the placental barrier. Embryonic and fetal tissues are considered at increased risk for DNA damage induced by xenobiotics since their cells are in rapid proliferation and differentiation. Determination of micronucleus (MN) frquencies has been shown to be useful for the evaluation of structural and chromosomal segregation damage. Its use in human monitoring of children and adults provides a sensitive and trustworthy biomarker for the assessment of DNA damage [1–4]. Even though it is feasible to evaluate MN in lymphocytes from umbilical cord blood it is striking that only three reports have been published on this subject, two being from the same group, and only one that used the cytokinesis-block methodology [5–7]. One of these reports evaluated the basal MN frequency in 28 newborns (NB) samples finding an average and range of 3.03 (0.5–9.5) in 1000 mononucleated cells [5]. The second study evaluated MN frequency in 19 samples, 9 from babies exposed in utero to ultrasonographic diagnostic procedures, and 10 from non-exposed NB. Averages and ranges reported were 3.4 (1–9.5) and 2.9 (0.5–8.6) in 1000 mononucleated cells, respectively, [6]. In the third study, micronucleated lymphocytes were scored in 18 NB cord bloods, observing a highest frequency of three micronucleated cells per 2000 binucleated cells [7]. The main objective of this research was to evaluate the frequency of MN in cells from umbilical cord blood as a potential assay for in utero exposure to genotoxic agents. NB belonged to one of the four groups: two groups of healthy babies born in two different cities, a third group environmentally exposed to pesticides, and a fourth group whose mothers presented high-risk pregnancies.
2. Materials and methods Mothers were invited to participate, and enrolled only if they signed an informed consent after the explanation of the objectives and stages of this study. Clinical characteristics, reproductive history, pesticide exposure, and smoking were evaluated with a structured questionnaire. After birth, venous blood samples were collected immediately from each NB umbilical cord, and maternal venous blood was obtained from two of the four groups studied.
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The characteristics of the four NB groups are: Group I. Twenty-one healthy NB from Chihuahua City, Mexico. Pregnancies developed without complications, and explicit occupational, environmental or home exposure to toxic compounds was denied in the questionnaire. Group II. Twelve healthy NB from Mexico city. These pregnancies were without complications and the mothers reported no occupational, environmental or home exposure to toxic compounds. Group III. Sixteen NB whose mothers lived in an agricultural zone in Delicias, Chihuahua in the North of Mexico. The mothers of these NB resided in an area exposed to pesticide mixtures (mainly organophosphates) during the summer and autumn spraying cycles. Group IV. Fifteen NB from Mexico city. Mothers gave birth in a hospital for women with highrisk pregnancies. Information about diseases and treatments was obtained from hospital registers. Blood samples were also obtained from mothers from groups I and III (17 and 14 samples, respectively). MN frequency was determined in both mothers and children in these two groups. Acetylcholinesterase concentration (AChE) was measured in groups I and III, to identify the enzyme concentration differences due to organophosphate pesticide exposure using the method of Ellman et al. [8] modified following the suggestions made by the World Health Organization [9]. A reduction between 20–30% in the enzyme activity evidence pesticide exposure [10]. Newborns were classified as having intrauterine growth retardation when they had a weight lower than the 10th percentile for the gestational week according to the tables of Overpeck et al. [11], and as a preterm NB when their gestational age was 0.05). Other studies in women 20–30 years old have reported simi-
lar (3.04 ± 2.4) [17], or higher frequencies (7.4 ± 0.75) [18] and (6.79 ± 0.69) [19]. The correlation between the frequency of MN in mothers and their NB was statistically significant (r = 0.61, p < 0.01). We evaluated several factors that could modify the frequency of MN in NB. We considered mother’s age older than 30 years, retarded intrauterine growth, and preterm childbirth as potential modifiers for all groups. Table 2 shows the clinical characteristics of both mothers and NB. A significant difference in weight and size was observed in groups II and IV when compared to group I. Smoking during pregnancy was denied by mothers from groups I and II, and only one and two mothers from groups III and IV, respectively, acknowledged a tobacco habit. Fifty-one percent of mothers worked at home, 34% were employed in cloth manufacturing or commerce, 15% were professionals, and
Table 2 Clinical characteristics of mothers and newborns Variable
Group I X ± S.D.a n = 21
Mothers age (years) Weeks of gestation Pregnancy number Prenatal c consultations Newborn weight (g) Newborn size (cm) Maternal AChE (U/mL)c Newborn AChE (U/mL)
25 39 2 6 3356 51 5.77 4
± ± ± ± ± ± ± ±
6 1 1 2 467 3 0.9 1.07
Group II X ± S.D.a n = 12
Group III X ± S.D.a n = 16
27 ± 39 ± 2± 6± 3062 ± 49 ± N.D. N.D.
24 40 2 7 3241 52 5.73 3.98
6 1 1 2 273b 1b
N.D.: not determined. a Mean ± standard deviation. b p < 0.05. c Blood samples taken from mothers from groups I and III (17 and 14 samples, respectively). d p > 0.05.
± ± ± ± ± ± ± ±
4 1b 1 2 394 2 1.6d 0.68d
Group IV X ± S.D.a n = 15 28 ± 38 ± 2± 7± 3022 ± 48 ± N.D. N.D.
7 1b 1 3 409b 3b
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only one from group IV was recognized to be occupationally exposed to solvents. However, no significant difference in the frequency of MN in NB cells, which could be linked to any of these variables was observed between groups (p > 0.05). Concerning NB’s gender (34 males and 30 females), in our study (groups I–III) MN frequency was similar for male and female NB (1.52 ± 1.26 versus 2.37 ± 2.06); and we only observed a higher frequency of MN in female NB from group IV (4.5 ± 2.06) compared to male NB from the same group (1.75 ± 0.75, p = 0.02). Other researchers have not found a significant variation in the frequency of MN among NB of different sex, and reported that sex was an important determinant for MN variation only after the forth decade of life [14]. AChE levels were also determined both in mothers and in children from groups I and III to assess exposure to pesticides. The levels found in samples obtained from both groups were similar. A positive correlation between AChE values from mothers and NB was found r = 0.63 (p < 0.01). NB had significantly lower AChE activity than their mothers (p < 0.01). Similar results have been reported in other studies [20,21]. There was no significant difference in the MN frequency of newborns whose mothers had a normal AChE activity versus the ones whose mothers had a 20% or more reduced AChE activity (1.6 ± 0.92 versus 1.3 ± 1.3) (p = 0.24). In addition, there was no significant difference in the MN frequency in mothers from groups I and III. Perhaps environmental exposure to pesticides in women from group III was low and therefore not enough to produce genotoxic damage in them or their babies. The fact that we did not find differences in AChE levels would support the low exposure mentioned; nevertheless, in a study carried out in 450 individuals in the same area in a different period, a 20% decrease in AChE activity was found in 97 cases [22]. It should be mentioned that sample size and seasonal variations in the use of particular pesticides might also be playing a role in the different results obtained in both studies. It has been pointed out that age might be one of the factors implicated in the genotoxic effect of pesticides [23], and since this study was performed with young women, it could also explain the absence of an increased MN frequency. Cytogenetics monitoring of individuals exposed to pesticides (farmers, floriculture workers and fumigators) has shown increased genotoxic damage when compared with non-exposed groups [23,24]. Other
authors have not found differences [25–27]. This controversy could reflect different amounts of pesticide exposure, the genotoxic potential of the specific pesticide, the use of protection measures [3], or even genetic differences in the sensitivity to the specific pesticide due to polymorphisms in the genes involved in their metabolism. In this respect, when comparing the MN frequency distribution in each of the four groups, it was observed that both group III (NB from mothers exposed to pesticides during pregnancy) and IV (NB from mothers with a high-risk pregnancy), present more individuals with a higher MN frequency than groups I and II (Fig. 2). This could indicate that there were some individuals in group III either more exposed or more sensitive to the effects of pesticides, while the higher frequency in group IV is probably due to the complicated course of pregnancy or to the medical treatments received. Medications prescribed to mothers with high-risk pregnancies, as well as the weight and MN number found in their NB are described in Table 3. Since this was a random sample, a direct correlation was not possible between the MN frequency and some medication prescribed to the patients. Data, however, suggest that several factors may interact to influence DNA damage in NB. Appropriate studies should be carried out to determine if specific medications prescribed during pregnancy (for example, anticonvulsants) produce genotoxic effects in NB. The fetus may be exposed to multiple xenobiotics during pregnancy, which can be involved in the development of diseases in later life. Therefore, it is important to have methods to identify potential transplacental mutagens. Umbilical cord blood cells have been used to evaluate some endpoints that measure DNA damage, such as HPRT mutations [28] and chromosomal aberrations [29]. In the present study we determined that evaluation of MN in umbilical cord lymphocytes is a simple method that may be added to the battery of tests to assess DNA damaging activity, and since it is possible to differentiate MN generated by DNA breaks from those resulting from chromosomal mal-segregation it represents a unique method to identify both clastogens and aneugens. We expect that the current results will prompt further research in this direction.
M. Levario-Carrillo et al. / Mutation Research 586 (2005) 68–75
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Fig. 2. Frequencies distribution of micronucleated lymphocytes in newborns. Groups I and II represent the newborns from two urban cities. Group III represents newborns from an agricultural area. Group IV includes newborns from high-risk pregnancies. Figure shows an increase in the number of individuals with higher MN frequencies in groups III and IV.
Table 3 Pregnancy condition of mothers in group IV and their newborns weight and micronucleus frequency Age of the mother (years)
Pregnancy (weeks)
Condition
Treatment
N.B.a weight (g)
MN/1000 BNCb N.B.
16 18 26 22 37 38 31
38 38 37 40 38 38 38
– – – – – – Ovulation inducers
3350 2770 2560 2860 3560 3400 2820
2 1 5 3 6 1 3
19 20
38 39
Phenytoin, fraxiparine Valproic acid
2780 3670
8 5
20 26 27 31 40 31
38 39 41 38 38 38
Young age Unknown Placenta previa Unknown Uterine myomatosis Unknown Myomectomy, infertility, induced- ovulation pregnancy Cerebrovascular event Gestational diabetes, epilepsy, psychomotor retardation Unknown Systemic lupus erythematous, vasculitis Hypothyroidism Gestational Diabetes hypothyroidism Exposure to paint thinner Pyelonephritis, infertility, cervicovaginitis
– Prednisone Thyroxin Thyroxin
3000 2925 3480 3290 2350 2528
8 7 2 2 4 3
a b
N.B.: newborn. BNC: binucleated cell.
Metamizol
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M. Levario-Carrillo et al. / Mutation Research 586 (2005) 68–75
Acknowledgments This study was partly supported by DGAPA (UNAM) and CONACYT. We thank Dr. Luis A. Herrera for the critical review of this manuscript.
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