No effect of maternal smoking in early pregnancy observed on chromosome aberrations in chorionic villus samples

Mutation Research, 298 (1993) 285-289

285

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00

MUTGEN 01864

No effect of maternal smoking in early pregnancy observed on chromosome aberrations in chorionic villus samples Kirsi Salonen

a

and Jaana L~ihdetie b

a Department of Genetics and b Department of Medical Genetics, University of Turku, SF-20520 Turku, Finland

(Received 19 June 1992) (Revision received 8 October 1992) (Accepted 15 October 1992)

Keywords: Chromosome aberrations; Chorionic villus samples; Smoking

Summary The effect of maternal smoking on first trimester chorionic villus samples (CVS) was studied by analysing the frequency of chromosome aberrations (CAs) among 20 non-smoking and 20 smoking mothers. The aberrations were classified as chromosome- and chromatid-type breaks and gaps. No statistically significant differences were found in the frequencies of CAs between non-smoking mothers (5.4% or 2.0% gaps excluded) and smoking mothers (3.5% or 1.0% gaps excluded).

Maternal smoking is a common harmful exposure during pregnancy and causes significant public health concern. An increase of chromosomal damage and somatic mutations has been observed in peripheral blood lymphocytes of smokers (IARC, 1985; Nordic Study Group, 1990; Tates et al., 1991a) and a broad spectrum of adverse effects on the foetus exposed to maternal smoking has been well documented, including intrauterine growth retardation, an increase in perinatal mortality and premature births (National Research Council, 1986; Surgeon General, 1986). The risk of these effects is further increased if the mother is 35 years old or older (Cnattingius, 1988; Wen et al., 1990).

Correspondence: Dr. J. L~ihdetie, Department of Medical Genetics, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku, Finland.

This study was designed to clarify whether cigarette smoking in the first trimester of pregnancy affects the frequency of chromosome aberrations (CAs) in trophoblast cells in chorionic villus samples (CVS). These cells have the same chromosome complement as the foetus itself. The frequencies of CAs (with and without gaps) was analysed in cells of semidirect preparations of 20 smoking and 20 non-smoking mothers. Materials and methods

Subjects The subjects were pregnant women who came to the Turku (TYKS) or Helsinki (HYKS) University Central Hospitals for the prenatal diagnosis of foetal karyotype because of advanced maternal age (37 years or over) or an earlier child with trisomy and had chorionic villus biopsy taken in the first trimester. They were asked to com-

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287 smokers were excluded) and the samples were processed similarly. Written informed consent was obtained from all the subjects of this study.

TABLE 2 FREQUENCIES OF CHROMOSOME ABERRATIONS IN CVS OF SMOKERS AND NON-SMOKERS

Semidirect chromosome preparations of CVS

Smokers

Non-smokers

20 1892

20 1944

3.5 2.0-5.0 1.0-12.0

5.4 p=0.11 3.1-7.0 1.0-11.0

1.0 0.0-2.0 0.0-5.0

2.0 p = 0.10 0.5-3.0 0.0-5.8

CVS was performed by either the transabdominal or the transcervical route in the first trimester of pregnancy. Villi cleaned from maternal tissue were processed following the technique of Simoni et al. (1983) with slight modifications. Briefly, after overnight incubation in Chang culture medium villi were incubated with colcemid at 0.06 t z g / m l for 2 h, hypotonised in 0.07 M KCI for 20 min and fixed in 3 : 1 m e t h a n o l - a c e t i c acid. Air-dried slides were stained with Giemsa.

Number of women Number of cells CAs including gaps (%) median 25-75% quantiles range CAs gaps excluded (%) median 25-75% quantiles range

Chromosome analysis

No increase in CAs was observed in CVSs of smoking mothers: 3.5% of cells had CAs or 1.0% if chromatid gaps were excluded. These differences are not statistically significant. Information on smoking habits was based on self-report by the mother and was not validated. It is possible that some pregnant smokers denied or underreported their smoking habits because of antismoking attitudes. T h r e e non-smokers may have had passive exposure to cigarette smoke due to their husbands smoking (Table 1). Biochemical techniques exist to measure cigarette consumption and passive smoking, e.g., m e a s u r e m e n t of serum thiocyanate (Hauth et al., 1984), serum or urine cotinine (Jarvis et al., 1984) or N-(2-hydroxyethyl)valine adducts in haemoglobin (Tates et al., 1991b, 1992) which might be used for more accurate exposure assessment. Eleven subjects reported usage of multivitamin or homoeotherapeutic agents, eight of them were non-smokers. Occupational exposures reported by five subjects were organic solvents, paints, glues and cleaning chemicals, and two smokers were regularly occupationally exposed to environmental tobacco smoke. It is difficult to evaluate the effects of these factors due to the limited number of individuals. Results on the effect of smoking on CAs of peripheral blood lymphocytes are contradictory (Hedner et al., 1983; Bigatti et al., 1985; Bender et al., 1988, 1989). It has been suggested that interindividual variation accounts for the lack of significant differences between smokers and

The coded slides were scored without knowledge of the smoking status. Only metaphases with good quality and with 46 + 1 chromosomes were analysed. Chromosome aberrations were grouped into chromatid and chromosome gaps (ctg and csg, respectively) and into chromatid and chromosome breaks (ctb and csb, respectively) according to the classification of the Nordic Study G r o u p (Brogger et al., 1984). Acentrics and marker chromosomes were scored as one chromosome break (csb). Exchange-type aberrations, such as tri- or quadriradials, were not observed. In most cases 100 metaphases were analysed (Table 1). The frequencies of CAs (with or without gaps) in the smoking and non-smoking groups were compared counting Wilcoxon scores (rank sums) and performing Kruskall-Wallis test with the SAS Statistical Program package (SAS/Institute Inc., 1987). Results and discussion Thirty-seven out of 309 (12.0%) interviewed CVS patients reported that they were current smokers. Samples with an abnormal karyotype (except one X X Y case, Table 1) or not suitable for scoring were excluded so that the final study groups consisted of 20 smokers and 20 selected controls. Individual results are shown in Table 1. T h e frequency of c h r o m o s o m e aberrations (median) in CVSs of non-smokers was 5.4% or 2.0% if chromatid gaps were excluded (Table 2).

288 non-smokers ( I A R C , 1985). Further, light smoking (fewer than 20 cigarettes per day) may cause only slight effects which are not observable because o f individual variation (SinuEs et al., 1990). Earlier cytogenetic studies of transplacental effects of maternal smoking have yielded conflicting results. Exposure of p r e g n a n t mice to tobacco smoke has b e e n r e p o r t e d to increase the n u m b e r of micronucleated polychromatic erythrocytes in foetal mouse liver (Balansky and Blagoeva, 1989). In h u m a n s no increase of micronucleated foetal liver ceils has b e e n observed, but an increased frequency of SCEs has b e e n reported if the m o t h e r smoked (Karube et al., 1989). N o differences in SCE frequencies in cord blood samples of n e o n a t e s of smoking and non-smoking m o t h e r s have b e e n observed (Ardito et al., 1980; Seshadri et al., 1982; Sorsa et al., 1989). O u r result is in accordance with the earlier reports showing nonsignificant differences due to smoking. D e m o n s t r a t i o n of increased cotinine levels and mutagenicity in second trimester amniotic fluid (Sorsa et al., 1989; Vainio et al., 1988) or at term (Rivrud et al., 1986) and D N A adducts in placentas of smokers associated with r e d u c e d foetal weight (Everson et al., 1988) suggest that the foetus is exposed to cigarette smoke constituents in utero. In the present study, the exposure time of the CVS cells scored, the cytotrophoblasts of the placenta, was about 10 weeks if the m o t h e r was a smoker. T h e subacute nature and low level of exposure, the majority of smokers smoking fewer than 15 cigarettes a day, and a considerable interindividual variation may partly explain the negative result of our study. W e think that our observation of no clastogenic effect by maternal smoking in CVS should be confirmed by an experimental design taking into account that cigarette smoke is a weak clastogen, i.e., a considerably larger sample size is needed, and the study should possibly include biochemical measurements of exposure.

Acknowledgements W e thank the staff of the Prenatal Diagnostic Units of T u r k u and Helsinki University Central Hospitals for samples. T h e assistance of the C h r o m o s o m e L a b o r a t o r y staff of the D e p a r t m e n t

of Medical Genetics, University of T u r k u is appreciated. H a n s Helenius is acknowledged for help with statistics. This study was financially supported by T u r k u University Foundation.

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