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Cytogenetic evaluation of pre-pregnancy smoking in maternal and newborn lymphocytes
European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 205–209
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Cytogenetic evaluation of pre-pregnancy smoking in maternal and newborn lymphocytes Dimitra E. Kareli a,*, Stamatia E. Pouliliou a, Ioannis H. Nikas b, Afrodite N. Psillaki b, Georgios C. Galazios b, Vassilios A. Liberis b, Theodore S. Lialiaris a a b
Laboratory of Genetics, Medical School, Demokrition University of Thrace, Alexandroupolis, Greece Department of Obstetrics and Gynaecology, Medical School, Demokrition University of Thrace, Alexandroupolis, Greece
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 December 2011 Received in revised form 21 May 2012 Accepted 26 July 2012
Objective: To study cytogenetic damage in order to estimate the effect of pre-pregnancy smoking on pregnant women and their foetuses. Study design: Lymphocyte cultures were obtained from peripheral blood of 20 women who quit smoking during pregnancy, and umbilical cord blood of their newborns at delivery. Cytogenetic analyses were performed for sister chromatid exchanges (SCEs), proliferation rate index (PRI) and mitotic index (MI) using the Fluorescence Plus Giemsa staining technique. Twenty non-smoking women and their newborns were evaluated as controls. CPT-11, a known antineoplastic, was used as a positive genotoxic agent in order to correlate non-smoking women with smoking women and reveal any underlying chromosome instability. Statistical evaluation of SCE frequencies, PRI and MI was based on independent samples t-test in order to estimate the effect of pre-pregnancy smoking on mothers and their newborns. Results: SCEs were induced in the cord blood lymphocytes of newborns whose mothers smoked before pregnancy when they were exposed to the mutagenic agent CPT-11 (p < 0.01). A similar increase in SCEs was observed in both non-smoking and smoking mothers exposed to CPT-11. Newborns in both groups had significantly lower SCE levels than their mothers (p < 0.01). Conclusion: Pre-pregnancy smoking results in cytogenetic damage for both mothers and newborns, and is an important risk factor for cancer and/or other genetic-related diseases. Smoking cessation needs to occur well before conception in order to avoid the strong cytogenetic association between pre-pregnancy smoking by mothers and their newborns. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Smoking before pregnancy Umbilical cord blood Genotoxicity Cytostaticity Cytotoxicity
1. Introduction Tobacco smoke contains approximately 4700 chemicals, many of which are carcinogenic [1,2]. Tobacco use is the leading cause of preventable death, estimated to cause more than 5 million deaths each year worldwide [3]. Most people correlate smoking with lung cancer, but smoking is also associated with cancer of the oral and nasal cavities, oesophagus, larynx, pharynx, pancreas, liver, kidney, stomach, urinary tract and cervix. The carcinogens in tobacco smoke exert their biological effects through interaction of reactive intermediates with DNA to form DNA adducts. These substances induce oxidative DNA damage, and the mechanism by which smoking-related cancer is initiated includes exposure of human tissues and organs to these carcinogens, and consequent metabolic activation [1,4,5].
* Corresponding author at: Department of Genetics, Medical School, Demokrition University of Thrace, Alexandroupolis 68100, Greece. Tel.: +30 25510 30522; fax: +30 25510 30522. E-mail address: [email protected] (D.E. Kareli). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.07.035
Cigarette smoke is genotoxic in nearly all systems in which it has been tested. In rodents, smoke components induce sister chromatid exchanges (SCEs) and micronuclei in bone marrow and lung cells [5,6]. In humans, smoking reduces female fecundity and has toxic effects in the reproductive system, such as mutagenic effects in cervical mucus, induction of micronuclei in cervical epithelial cells, and genotoxic effects in amniotic fluid [5,7–9]. Sperm cells of male smokers show DNA adducts, strand breaks and oxidative damage, as well as increased aneuploidy. All of these data lead to the conclusion that tobacco smoke is a human germ-cell mutagen [5,9]. Unfortunately, tobacco is one of the most common substances used during pregnancy. Although in-utero exposure to tobacco smoke has perinatal and postnatal health consequences, almost 20–30% of women continue to smoke during pregnancy [3,10]. There is indirect evidence that certain tobacco metabolites can cross the placental barrier and become genotoxic to the foetus [9,11]. Prenatal tobacco exposure is associated with speech processing problems, levels of irritability and hypertonicity, attention levels and ability to self-regulate [12,13]. Pregnant
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women exposed to tobacco smoke appear to be a greater risk of DNA damage, including DNA strand breaks, SCEs, cell transformation and cytotoxicity, compared with non-exposed pregnant women [13–16]. Newborns of smoking mothers have elevated frequencies of HPRT mutants [17], DNA strand breaks, chromosomal losses [5,15] and DNA adducts [18], as well as greater risk of developing cancer in later life [11]. Moreover, newborns of mothers who smoked during pregnancy have a significantly higher frequency of translocations compared with newborns of non-smoking mothers [19]. Chromosomal fragility is a condition where chromosomes tend to form breaks, gaps and interchanges when the cell is exposed to partial replication stress. It is caused by several natural, chemical (DNA cross-linking agents) or biological factors, and occurs in different places in one or more chromosomes of one or more cells in increased levels compared with normal cells [20,21]. Chromosome instability diseases include Fanconi anaemia, Bloom syndrome, ataxia telangiectasia, Werner syndrome, xeroderma pigmentosum, hyperparathyroidism and, more recently, Nijmegen breakage syndrome, asthma and chronic renal failure [22,23]. Chromosomal fragility can also be induced by biological, natural and chemical environmental mutation factors such as smoking, alcohol, caffeine, stress, diseases (viruses or genetic) and irradiation [24–29]. Three cytogenetic parameters were evaluated in lymphocyte cultures of peripheral blood of pregnant women and umbilical cord blood of their newborns: (1) SCE levels, (2) proliferation rate index (PRI) and (3) mitotic index (MI). An SCE is a natural process that is directly associated with DNA synthesis during the S phase of the cell cycle. It involves the exchange of genetic material between two sister chromatids before they separate during metaphase [30–32]. Increasing frequency of SCEs indicates the existence of greater damage in the DNA of the cell due to several factors. Therefore, the SCE method has been proposed as a very sensitive, simple and rapid quantitative method to detect mutagens and/or carcinogens [30]. To evaluate the possible genotoxicity of pre-pregnancy smoking and to correlate non-smoking women with smoking women, irinotecan (CPT-11), a known antineoplastic, was used as a positive control to produce high levels of SCEs. CPT-11 is a DNAdamaging agent and is used to reveal any underlying chromosome instability as it has a synergistic genotoxic effect. CPT-11 is a semisynthetic analogue of camptothecin-11, a natural alkaloid extracted from the plant Camptotheca acuminata of the Nyssaceae family [33,34]. It acts as an inhibitor of the nuclear enzyme topoisomerase I, and represents a promising target for the development of new cancer chemotherapeutic agents against a number of solid human tumours [35]. Induction of SCEs has been observed due to the genotoxic effect of CPT-11 [36]. The influence of pre-pregnancy smoking on foetal development and newborn health is not fully understood. This study estimated the cytogenetic effects of pre-pregnancy smoking on pregnant women and their foetuses.
General Hospital of Alexandroupolis (Greece). Women from Group B smoked approximately 14 cigarettes per day before pregnancy. Both groups consisted of women who had full-term pregnancies without complications. 2.2. Blood samples Cultures of maternal peripheral lymphocytes and umbilical cord blood lymphocytes were prepared in universal containers by adding 11 drops of whole blood to 5 ml of chromosome medium B (Biochrom KG, Berlin, Germany). These were incubated at 37 8C for 72 h. Cells were allowed to proliferate for at least two mitotic cycles in the presence of BrdU. The final concentration of BrdU was 5 mg/ml. A final concentration of CPT-11 of 50 ng/ml was added at the beginning of the culture period. After 70 h, 0.5 mg/ml of colcemide was added for 2 h, and cultures were harvested at the end of the incubation period. All cultures were kept in the dark to prevent or minimize photolysis of BrdU. The chromosome preparations were stained using a modified Fluorescence Plus Giemsa (FPG) technique [25,28]. Scoring was performed in a blind fashion, and cells were counted at the first mitotic division (where both chromatids were heavily stained, Fig. 1), at the second mitotic division (where one chromatid of each chromosome was heavily stained, Fig. 2), and at third and subsequent mitotic divisions (where part of both chromatids of each chromosome were lightly stained, Fig. 3). Mean SCE levels were only evaluated in suitable second division metaphases (20 metaphases), as it is only possible to observe and estimate them at this stage. In order to establish PRI, 200 cells were counted for each culture, and the following formula was used: PRI = (M1 + 2M2 + 3M3+)/N, where M1 is the percentage of cells at first division, M2 is the percentage of cells at second division, and M3+ is the percentage of cells at third and subsequent divisions, while N is the total number of cells counted (M1 + M2 + M3+). Moreover, the MIs for 2000 activated lymphocytes were determined for all cultures. 2.3. Statistical analysis In order to compare various treatments, logarithmic transformation of SCE values was performed using one-way analysis of variance and the Duncan test for pair-wise comparisons. MI and
2. Materials and methods 2.1. Patients The study protocol was verified by the University General Hospital of Alexandroupolis Ethic Committee. Heparinized blood samples were received from 20 non-smoking women (average age 27 years, Group A) and their newborns, and 20 women who quit smoking while they were pregnant (average age 30 years, Group B) and their newborns. Newborn samples were taken from umbilical cord blood at the time of delivery. All samples were obtained from the Department of Obstetrics and Gynaecology of the University
Fig. 1. A first-division metaphase after Fluorescence Plus Giemsa staining of a newborn umbilical cord blood lymphocyte, where both chromatids in all chromosomes are heavily stained. Original magnification 1000.
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compared with control cultures: 33.98 SCEs/cell in Group A and 38.92 SCEs/cell in Group B. PRI, which is a qualitative index of cytostaticity, generally showed a strong but not significant decrease. The exception was for newborns from Group A, for whom a significant decrease in CPT-11 cultures was found compared with control cultures. For MI, which is a qualitative index of cytotoxicity, a significant decrease was found in the control cultures of mothers compared with their newborns in both groups (p < 0.05); however, only Group A showed a significant decrease in the CPT-11 cultures (p < 0.05). 3.1. Comments
Fig. 2. A second-division metaphase after Fluorescence Plus Giemsa staining of a newborn umbilical cord blood lymphocyte, where all chromosomes have one lightly stained chromatid and one heavily stained chromatid (arrows show sister chromatid exchanges). Original magnification 1000.
PRI were evaluated using Chi-squared test. A p-value < 0.05 was considered to indicate statistical significance.
3. Results Table 1 shows the findings for both mothers and newborns. There were no significant differences in SCE levels, which is a quantitative index of genotoxicity, between mothers from Group A (4.40 SCEs/cell) and mothers from Group B (5.00 SCEs/cell). Similar results were also found for the newborns in both groups: 3.78 SCEs/cell and 3.90 SCEs/cell, respectively. Newborns in both groups had significantly lower SCE levels than their mothers (p < 0.01). Addition of the mutagenic agent CPT-11 led to a significant increase in SCEs in newborns compared with their mothers in both groups (p < 0.01). Mothers exposed to CPT-11 had a significant increase (p < 0.01) in SCE levels compared with control cultures: 24.29 SCEs/cell in Group A and 28.84 SCEs/cell in Group B. A similar increase was also observed in newborns exposed to CPT-11
Fig. 3. A third- (or subsequent) division metaphase after Fluorescence Plus Giemsa staining of a newborn umbilical cord blood lymphocyte, where some chromosomes have two lightly stained chromatids. Original magnification 1000.
Increasing frequency of SCEs indicates the existence of genotoxicity and therefore increasing DNA damage in cells due to several factors. This damage indicates the failure of prereplicative repair mechanisms to achieve full recovery of the damaged site, thus leading to double-strand breaks. Therefore, the SCE method has been proposed as a very sensitive, simple and rapid method for detecting mutagens and/or carcinogens [31]. Tobacco smoke is a mutagen as it is genotoxic in nearly in all systems in which it has been tested [5]. Both pregnant women who had been exposed to tobacco smoke and their newborns were at higher risk of DNA damage (DNA strand breaks, SCEs, cell transformation, cytotoxicity, chromosomal losses, DNA adducts) compared with non-smoking mothers and their newborns [5,14–18]. This study evaluated the cytogenetic effects of pre-pregnancy smoking on pregnant women and their foetuses. The results indicate that the DNA of newborns whose mothers smoked before pregnancy had chromosome instability, shown by increased SCE levels in umbilical cord blood lymphocytes exposed to CPT-11 (Table 1). Similar results were observed in mothers exposed to CPT-11, as Group B had higher SCE levels than Group A. In the absence of CPT-11, the SCE levels of newborns were lower than those of their mothers in both groups. However, addition of the mutagenic agent reversed these results by increasing the SCEs/cell in newborns. The finding that the SCE frequency in newborns was significantly lower than that of their mothers in both smoking and non-smoking groups is consistent with data from other studies [37–41]. The present authors agree with Sardas et al. that the difference between adults and newborns seems to reflect the difference in exposure, as newborns had no direct contact with DNA-damaging agents [40]. Furthermore, the above observation was confirmed from the study data, as addition of the mutagenic agent CPT-11 led to a significant increase in the SCE levels of newborns, not only compared with their mothers but also between the two study groups. It seems that smoking can influence this sensitive indicator of genotoxicity (SCEs) when foetuses are exposed to a DNA-damaging agent. It is known that tobacco metabolites can cross the placental barrier and become genotoxic to the foetus [14]. Cord serum cotinine has been proposed as the most adequate biomarker of foetal exposure to smoking at the end of pregnancy. Studies have found a strong association between maternal and newborn plasma cotinine concentrations, concluding that there is probably no placental barrier for plasma cotinine between pregnant mothers and their newborns. This lack of placental barrier for cotinine (and probably for nicotine) can partially explain smoking-related perinatal disorders [1,42–44]. Given that the smoking group in the present study consisted of mothers who quit smoking during pregnancy could lead to speculation. However, there was a lapse of time between confirmation of pregnancy and smoking cessation, and this period corresponds with the early stages of development; as such, these results are indicative of DNA damage caused in this sensitive stage
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Table 1 Effect of smoking before pregnancy in maternal and umbilical cord blood lymphocytes. Cytogenetic indices from 20 non-smoking mothers and their newborns Mean SCEs/cell
(1) (2) (3) (4)
Mothers, control Mothers, CPT-11 Newborns, control Newborns, CPT-11
Proliferation rate index
Mitotic index (%)
SCEs SEM (diff control-CPT-11)
No. of metaphases counted per donor: 30
PRI
No. of metaphases counted per donor: 200
MI
No. of interphase nuclei counted per donor: 2000
4.40 0.16a 24.29 0.74b (20.89) 3.78 0.16 33.98 1.03c (30.20)
Total: Total: Total: Total:
2.21 2.16 2.33f 2.10
Total: Total: Total: Total:
12.95g 10.40 19.40 15.25
Total: Total: Total: Total:
600 600 600 600
4000 4000 4000 4000
40,000 40,000 40,000 40,000
Cytogenetic indices from 20 smoking mothers who quit smoking during pregnancy and their newborns Mean SCEs/cell
(5) (6) (7) (8)
Mothers, control Mothers, CPT-11 Newborns, control Newborns, CPT-11
Proliferation rate index
Mitotic index (%)
SCEs SEM (diff control-CPT-11)
No. of metaphases counted per donor: 30
PRI
No. of metaphases counted per donor: 200
MI
No. of interphase nuclei counted per donor: 2000
5.00 0.20d 28.84 0.58 (23.84) 3.90 0.14e 38.92 1.08 (35.02)
Total: Total: Total: Total:
2.09 2.09 2.35 2.21
Total: Total: Total: Total:
13.55h 12.05i 19.70 16.25
Total: Total: Total: Total:
600 600 600 600
4000 4000 4000 4000
40,000 40,000 40,000 40,000
SCEs, sister chromatid exchanges; PRI, proliferation rate index; MI, mitotic index; CPT-11, irinotecan; SEM, standard error of the mean; diff, difference. a p < 0.01 vs. lines 2–4. b p < 0.01 vs. lines 4 and 6. c p < 0.01 vs. line 8. d p < 0.01 vs. lines 6–8. e p < 0.01 vs. lines 5 and 8. f p < 0.05 vs. line 4. g p < 0.05 vs. line 3. h p < 0.05 vs. line 7. i p < 0.05 vs. line 8.
of pregnancy. This is consistent with Shulman et al. who found elevated frequencies of SCEs in directly prepared cytotrophoblasts obtained from women who smoked, concluding that there is direct placental DNA damage which could explain the deleterious effects of smoking during pregnancy [45]. For PRI, addition of the mutagenic agent led to a strong but not significant decrease (cytostaticity) in nearly all cultures. Nevertheless, a significant decrease was seen in newborns in Group A exposed to the mutagenic agent. The control cultures (not exposed to CPT-11) confirmed the increased proliferation capabilities of umbilical cord blood lymphocytes in both groups, as control cultures of newborns had higher PRI values than CPT-11 cultures. Finally, the results indicate that smoking during pregnancy induces cytotoxicity, reflected by the decreased MI of umbilical cord blood lymphocytes in control cultures of newborns from Groups A and B. On the other hand, the high survival potency of newborns’ lymphocytes was verified by the greater cytotoxic action of CPT-11 on maternal lymphocytes compared with their newborns. Tobacco is one of the most common substances used during pregnancy. It contains many carcinogens but its influence on foetal development and newborn health is not fully understood. This knowledge gap also includes cytogenetic damage of foetuses whose mothers smoke before pregnancy and quit after confirmation of pregnancy. Almost 20–30% of women continue to smoke during pregnancy [3,6,10], but a considerable number of women quit smoking during pregnancy in order to improve the health and development of their foetus. However, some pregnancies are not planned, and women continue to smoke because they are not aware of their situation (<3 months of gestation). These women were considered in the present study in terms of the cytogenetic damage that they might induce to their children through smoking. Moreover, to the authors’ knowledge, this is the first study to investigate the cytogenetic effect of pre-pregnancy smoking using the mutagenic agent CPT-11. Increasing frequency of SCEs indicates the existence of genotoxicity, and therefore increasing DNA damage in cells due
to several mutagenic factors [9,31]. The present results show that the DNA of newborns whose mothers smoked before pregnancy had chromosome instability, as shown by the increased SCE levels of umbilical cord blood lymphocytes exposed to CPT-11. This indicates that DNA damage was caused in the early stages of development, such as the embryonic stage. Obviously, it is not possible to control all cases of pregnancy, but women who are planning to have children should be advised to quit smoking well before conception in order to improve the health of their foetus. It has been known for many years that certain tobacco metabolites can cross the placenta and become toxic to the foetus [18,42–44]. It seems that smoking cessation in the early stages of pregnancy is not sufficient to avoid this. Apart from the direct adverse effects of smoking, there is also indirect damage such as cytogenetic damage, reflected by the increased frequency of SCEs. This type of damage can act synergistically with other environmental and/or chemical mutagenic substances to which people are exposed, willingly or otherwise, leading to carcinogenicity [13]. Observations of daily life raise concern that tobacco, alcohol and drugs are increasingly pressing problems, including among young/ pregnant women. This is both a medical and a socio-economic issue. In conclusion, the data indicate a strong association between maternal smoking before pregnancy and their newborns. The DNA of newborns whose mothers smoked before pregnancy appeared to have chromosome instability, shown by higher levels of SCEs in umbilical cord blood lymphocytes exposed to CPT-11. However, the increased proliferation capabilities and high survival potency of umbilical cord blood lymphocytes in control cultures (not exposed to CPT-11) suggest that these cells are dynamic. The genotoxic effects of smoking can be considered as important risk factors for cancer and/or genetic-related diseases. It appears that smoking cessation in early pregnancy is not sufficient, so it is strongly recommended that smoking cessation programmes should re-evaluate existing results and consider new directions for women planning to have children. The authors believe that smoking cessation needs to occur well before conception in order
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European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Cytogenetic evaluation of pre-pregnancy smoking in maternal and newborn lymphocytes Dimitra E. Kareli a,*, Stamatia E. Pouliliou a, Ioannis H. Nikas b, Afrodite N. Psillaki b, Georgios C. Galazios b, Vassilios A. Liberis b, Theodore S. Lialiaris a a b
Laboratory of Genetics, Medical School, Demokrition University of Thrace, Alexandroupolis, Greece Department of Obstetrics and Gynaecology, Medical School, Demokrition University of Thrace, Alexandroupolis, Greece
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 December 2011 Received in revised form 21 May 2012 Accepted 26 July 2012
Objective: To study cytogenetic damage in order to estimate the effect of pre-pregnancy smoking on pregnant women and their foetuses. Study design: Lymphocyte cultures were obtained from peripheral blood of 20 women who quit smoking during pregnancy, and umbilical cord blood of their newborns at delivery. Cytogenetic analyses were performed for sister chromatid exchanges (SCEs), proliferation rate index (PRI) and mitotic index (MI) using the Fluorescence Plus Giemsa staining technique. Twenty non-smoking women and their newborns were evaluated as controls. CPT-11, a known antineoplastic, was used as a positive genotoxic agent in order to correlate non-smoking women with smoking women and reveal any underlying chromosome instability. Statistical evaluation of SCE frequencies, PRI and MI was based on independent samples t-test in order to estimate the effect of pre-pregnancy smoking on mothers and their newborns. Results: SCEs were induced in the cord blood lymphocytes of newborns whose mothers smoked before pregnancy when they were exposed to the mutagenic agent CPT-11 (p < 0.01). A similar increase in SCEs was observed in both non-smoking and smoking mothers exposed to CPT-11. Newborns in both groups had significantly lower SCE levels than their mothers (p < 0.01). Conclusion: Pre-pregnancy smoking results in cytogenetic damage for both mothers and newborns, and is an important risk factor for cancer and/or other genetic-related diseases. Smoking cessation needs to occur well before conception in order to avoid the strong cytogenetic association between pre-pregnancy smoking by mothers and their newborns. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Smoking before pregnancy Umbilical cord blood Genotoxicity Cytostaticity Cytotoxicity
1. Introduction Tobacco smoke contains approximately 4700 chemicals, many of which are carcinogenic [1,2]. Tobacco use is the leading cause of preventable death, estimated to cause more than 5 million deaths each year worldwide [3]. Most people correlate smoking with lung cancer, but smoking is also associated with cancer of the oral and nasal cavities, oesophagus, larynx, pharynx, pancreas, liver, kidney, stomach, urinary tract and cervix. The carcinogens in tobacco smoke exert their biological effects through interaction of reactive intermediates with DNA to form DNA adducts. These substances induce oxidative DNA damage, and the mechanism by which smoking-related cancer is initiated includes exposure of human tissues and organs to these carcinogens, and consequent metabolic activation [1,4,5].
* Corresponding author at: Department of Genetics, Medical School, Demokrition University of Thrace, Alexandroupolis 68100, Greece. Tel.: +30 25510 30522; fax: +30 25510 30522. E-mail address: [email protected] (D.E. Kareli). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.07.035
Cigarette smoke is genotoxic in nearly all systems in which it has been tested. In rodents, smoke components induce sister chromatid exchanges (SCEs) and micronuclei in bone marrow and lung cells [5,6]. In humans, smoking reduces female fecundity and has toxic effects in the reproductive system, such as mutagenic effects in cervical mucus, induction of micronuclei in cervical epithelial cells, and genotoxic effects in amniotic fluid [5,7–9]. Sperm cells of male smokers show DNA adducts, strand breaks and oxidative damage, as well as increased aneuploidy. All of these data lead to the conclusion that tobacco smoke is a human germ-cell mutagen [5,9]. Unfortunately, tobacco is one of the most common substances used during pregnancy. Although in-utero exposure to tobacco smoke has perinatal and postnatal health consequences, almost 20–30% of women continue to smoke during pregnancy [3,10]. There is indirect evidence that certain tobacco metabolites can cross the placental barrier and become genotoxic to the foetus [9,11]. Prenatal tobacco exposure is associated with speech processing problems, levels of irritability and hypertonicity, attention levels and ability to self-regulate [12,13]. Pregnant
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women exposed to tobacco smoke appear to be a greater risk of DNA damage, including DNA strand breaks, SCEs, cell transformation and cytotoxicity, compared with non-exposed pregnant women [13–16]. Newborns of smoking mothers have elevated frequencies of HPRT mutants [17], DNA strand breaks, chromosomal losses [5,15] and DNA adducts [18], as well as greater risk of developing cancer in later life [11]. Moreover, newborns of mothers who smoked during pregnancy have a significantly higher frequency of translocations compared with newborns of non-smoking mothers [19]. Chromosomal fragility is a condition where chromosomes tend to form breaks, gaps and interchanges when the cell is exposed to partial replication stress. It is caused by several natural, chemical (DNA cross-linking agents) or biological factors, and occurs in different places in one or more chromosomes of one or more cells in increased levels compared with normal cells [20,21]. Chromosome instability diseases include Fanconi anaemia, Bloom syndrome, ataxia telangiectasia, Werner syndrome, xeroderma pigmentosum, hyperparathyroidism and, more recently, Nijmegen breakage syndrome, asthma and chronic renal failure [22,23]. Chromosomal fragility can also be induced by biological, natural and chemical environmental mutation factors such as smoking, alcohol, caffeine, stress, diseases (viruses or genetic) and irradiation [24–29]. Three cytogenetic parameters were evaluated in lymphocyte cultures of peripheral blood of pregnant women and umbilical cord blood of their newborns: (1) SCE levels, (2) proliferation rate index (PRI) and (3) mitotic index (MI). An SCE is a natural process that is directly associated with DNA synthesis during the S phase of the cell cycle. It involves the exchange of genetic material between two sister chromatids before they separate during metaphase [30–32]. Increasing frequency of SCEs indicates the existence of greater damage in the DNA of the cell due to several factors. Therefore, the SCE method has been proposed as a very sensitive, simple and rapid quantitative method to detect mutagens and/or carcinogens [30]. To evaluate the possible genotoxicity of pre-pregnancy smoking and to correlate non-smoking women with smoking women, irinotecan (CPT-11), a known antineoplastic, was used as a positive control to produce high levels of SCEs. CPT-11 is a DNAdamaging agent and is used to reveal any underlying chromosome instability as it has a synergistic genotoxic effect. CPT-11 is a semisynthetic analogue of camptothecin-11, a natural alkaloid extracted from the plant Camptotheca acuminata of the Nyssaceae family [33,34]. It acts as an inhibitor of the nuclear enzyme topoisomerase I, and represents a promising target for the development of new cancer chemotherapeutic agents against a number of solid human tumours [35]. Induction of SCEs has been observed due to the genotoxic effect of CPT-11 [36]. The influence of pre-pregnancy smoking on foetal development and newborn health is not fully understood. This study estimated the cytogenetic effects of pre-pregnancy smoking on pregnant women and their foetuses.
General Hospital of Alexandroupolis (Greece). Women from Group B smoked approximately 14 cigarettes per day before pregnancy. Both groups consisted of women who had full-term pregnancies without complications. 2.2. Blood samples Cultures of maternal peripheral lymphocytes and umbilical cord blood lymphocytes were prepared in universal containers by adding 11 drops of whole blood to 5 ml of chromosome medium B (Biochrom KG, Berlin, Germany). These were incubated at 37 8C for 72 h. Cells were allowed to proliferate for at least two mitotic cycles in the presence of BrdU. The final concentration of BrdU was 5 mg/ml. A final concentration of CPT-11 of 50 ng/ml was added at the beginning of the culture period. After 70 h, 0.5 mg/ml of colcemide was added for 2 h, and cultures were harvested at the end of the incubation period. All cultures were kept in the dark to prevent or minimize photolysis of BrdU. The chromosome preparations were stained using a modified Fluorescence Plus Giemsa (FPG) technique [25,28]. Scoring was performed in a blind fashion, and cells were counted at the first mitotic division (where both chromatids were heavily stained, Fig. 1), at the second mitotic division (where one chromatid of each chromosome was heavily stained, Fig. 2), and at third and subsequent mitotic divisions (where part of both chromatids of each chromosome were lightly stained, Fig. 3). Mean SCE levels were only evaluated in suitable second division metaphases (20 metaphases), as it is only possible to observe and estimate them at this stage. In order to establish PRI, 200 cells were counted for each culture, and the following formula was used: PRI = (M1 + 2M2 + 3M3+)/N, where M1 is the percentage of cells at first division, M2 is the percentage of cells at second division, and M3+ is the percentage of cells at third and subsequent divisions, while N is the total number of cells counted (M1 + M2 + M3+). Moreover, the MIs for 2000 activated lymphocytes were determined for all cultures. 2.3. Statistical analysis In order to compare various treatments, logarithmic transformation of SCE values was performed using one-way analysis of variance and the Duncan test for pair-wise comparisons. MI and
2. Materials and methods 2.1. Patients The study protocol was verified by the University General Hospital of Alexandroupolis Ethic Committee. Heparinized blood samples were received from 20 non-smoking women (average age 27 years, Group A) and their newborns, and 20 women who quit smoking while they were pregnant (average age 30 years, Group B) and their newborns. Newborn samples were taken from umbilical cord blood at the time of delivery. All samples were obtained from the Department of Obstetrics and Gynaecology of the University
Fig. 1. A first-division metaphase after Fluorescence Plus Giemsa staining of a newborn umbilical cord blood lymphocyte, where both chromatids in all chromosomes are heavily stained. Original magnification 1000.
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compared with control cultures: 33.98 SCEs/cell in Group A and 38.92 SCEs/cell in Group B. PRI, which is a qualitative index of cytostaticity, generally showed a strong but not significant decrease. The exception was for newborns from Group A, for whom a significant decrease in CPT-11 cultures was found compared with control cultures. For MI, which is a qualitative index of cytotoxicity, a significant decrease was found in the control cultures of mothers compared with their newborns in both groups (p < 0.05); however, only Group A showed a significant decrease in the CPT-11 cultures (p < 0.05). 3.1. Comments
Fig. 2. A second-division metaphase after Fluorescence Plus Giemsa staining of a newborn umbilical cord blood lymphocyte, where all chromosomes have one lightly stained chromatid and one heavily stained chromatid (arrows show sister chromatid exchanges). Original magnification 1000.
PRI were evaluated using Chi-squared test. A p-value < 0.05 was considered to indicate statistical significance.
3. Results Table 1 shows the findings for both mothers and newborns. There were no significant differences in SCE levels, which is a quantitative index of genotoxicity, between mothers from Group A (4.40 SCEs/cell) and mothers from Group B (5.00 SCEs/cell). Similar results were also found for the newborns in both groups: 3.78 SCEs/cell and 3.90 SCEs/cell, respectively. Newborns in both groups had significantly lower SCE levels than their mothers (p < 0.01). Addition of the mutagenic agent CPT-11 led to a significant increase in SCEs in newborns compared with their mothers in both groups (p < 0.01). Mothers exposed to CPT-11 had a significant increase (p < 0.01) in SCE levels compared with control cultures: 24.29 SCEs/cell in Group A and 28.84 SCEs/cell in Group B. A similar increase was also observed in newborns exposed to CPT-11
Fig. 3. A third- (or subsequent) division metaphase after Fluorescence Plus Giemsa staining of a newborn umbilical cord blood lymphocyte, where some chromosomes have two lightly stained chromatids. Original magnification 1000.
Increasing frequency of SCEs indicates the existence of genotoxicity and therefore increasing DNA damage in cells due to several factors. This damage indicates the failure of prereplicative repair mechanisms to achieve full recovery of the damaged site, thus leading to double-strand breaks. Therefore, the SCE method has been proposed as a very sensitive, simple and rapid method for detecting mutagens and/or carcinogens [31]. Tobacco smoke is a mutagen as it is genotoxic in nearly in all systems in which it has been tested [5]. Both pregnant women who had been exposed to tobacco smoke and their newborns were at higher risk of DNA damage (DNA strand breaks, SCEs, cell transformation, cytotoxicity, chromosomal losses, DNA adducts) compared with non-smoking mothers and their newborns [5,14–18]. This study evaluated the cytogenetic effects of pre-pregnancy smoking on pregnant women and their foetuses. The results indicate that the DNA of newborns whose mothers smoked before pregnancy had chromosome instability, shown by increased SCE levels in umbilical cord blood lymphocytes exposed to CPT-11 (Table 1). Similar results were observed in mothers exposed to CPT-11, as Group B had higher SCE levels than Group A. In the absence of CPT-11, the SCE levels of newborns were lower than those of their mothers in both groups. However, addition of the mutagenic agent reversed these results by increasing the SCEs/cell in newborns. The finding that the SCE frequency in newborns was significantly lower than that of their mothers in both smoking and non-smoking groups is consistent with data from other studies [37–41]. The present authors agree with Sardas et al. that the difference between adults and newborns seems to reflect the difference in exposure, as newborns had no direct contact with DNA-damaging agents [40]. Furthermore, the above observation was confirmed from the study data, as addition of the mutagenic agent CPT-11 led to a significant increase in the SCE levels of newborns, not only compared with their mothers but also between the two study groups. It seems that smoking can influence this sensitive indicator of genotoxicity (SCEs) when foetuses are exposed to a DNA-damaging agent. It is known that tobacco metabolites can cross the placental barrier and become genotoxic to the foetus [14]. Cord serum cotinine has been proposed as the most adequate biomarker of foetal exposure to smoking at the end of pregnancy. Studies have found a strong association between maternal and newborn plasma cotinine concentrations, concluding that there is probably no placental barrier for plasma cotinine between pregnant mothers and their newborns. This lack of placental barrier for cotinine (and probably for nicotine) can partially explain smoking-related perinatal disorders [1,42–44]. Given that the smoking group in the present study consisted of mothers who quit smoking during pregnancy could lead to speculation. However, there was a lapse of time between confirmation of pregnancy and smoking cessation, and this period corresponds with the early stages of development; as such, these results are indicative of DNA damage caused in this sensitive stage
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Table 1 Effect of smoking before pregnancy in maternal and umbilical cord blood lymphocytes. Cytogenetic indices from 20 non-smoking mothers and their newborns Mean SCEs/cell
(1) (2) (3) (4)
Mothers, control Mothers, CPT-11 Newborns, control Newborns, CPT-11
Proliferation rate index
Mitotic index (%)
SCEs SEM (diff control-CPT-11)
No. of metaphases counted per donor: 30
PRI
No. of metaphases counted per donor: 200
MI
No. of interphase nuclei counted per donor: 2000
4.40 0.16a 24.29 0.74b (20.89) 3.78 0.16 33.98 1.03c (30.20)
Total: Total: Total: Total:
2.21 2.16 2.33f 2.10
Total: Total: Total: Total:
12.95g 10.40 19.40 15.25
Total: Total: Total: Total:
600 600 600 600
4000 4000 4000 4000
40,000 40,000 40,000 40,000
Cytogenetic indices from 20 smoking mothers who quit smoking during pregnancy and their newborns Mean SCEs/cell
(5) (6) (7) (8)
Mothers, control Mothers, CPT-11 Newborns, control Newborns, CPT-11
Proliferation rate index
Mitotic index (%)
SCEs SEM (diff control-CPT-11)
No. of metaphases counted per donor: 30
PRI
No. of metaphases counted per donor: 200
MI
No. of interphase nuclei counted per donor: 2000
5.00 0.20d 28.84 0.58 (23.84) 3.90 0.14e 38.92 1.08 (35.02)
Total: Total: Total: Total:
2.09 2.09 2.35 2.21
Total: Total: Total: Total:
13.55h 12.05i 19.70 16.25
Total: Total: Total: Total:
600 600 600 600
4000 4000 4000 4000
40,000 40,000 40,000 40,000
SCEs, sister chromatid exchanges; PRI, proliferation rate index; MI, mitotic index; CPT-11, irinotecan; SEM, standard error of the mean; diff, difference. a p < 0.01 vs. lines 2–4. b p < 0.01 vs. lines 4 and 6. c p < 0.01 vs. line 8. d p < 0.01 vs. lines 6–8. e p < 0.01 vs. lines 5 and 8. f p < 0.05 vs. line 4. g p < 0.05 vs. line 3. h p < 0.05 vs. line 7. i p < 0.05 vs. line 8.
of pregnancy. This is consistent with Shulman et al. who found elevated frequencies of SCEs in directly prepared cytotrophoblasts obtained from women who smoked, concluding that there is direct placental DNA damage which could explain the deleterious effects of smoking during pregnancy [45]. For PRI, addition of the mutagenic agent led to a strong but not significant decrease (cytostaticity) in nearly all cultures. Nevertheless, a significant decrease was seen in newborns in Group A exposed to the mutagenic agent. The control cultures (not exposed to CPT-11) confirmed the increased proliferation capabilities of umbilical cord blood lymphocytes in both groups, as control cultures of newborns had higher PRI values than CPT-11 cultures. Finally, the results indicate that smoking during pregnancy induces cytotoxicity, reflected by the decreased MI of umbilical cord blood lymphocytes in control cultures of newborns from Groups A and B. On the other hand, the high survival potency of newborns’ lymphocytes was verified by the greater cytotoxic action of CPT-11 on maternal lymphocytes compared with their newborns. Tobacco is one of the most common substances used during pregnancy. It contains many carcinogens but its influence on foetal development and newborn health is not fully understood. This knowledge gap also includes cytogenetic damage of foetuses whose mothers smoke before pregnancy and quit after confirmation of pregnancy. Almost 20–30% of women continue to smoke during pregnancy [3,6,10], but a considerable number of women quit smoking during pregnancy in order to improve the health and development of their foetus. However, some pregnancies are not planned, and women continue to smoke because they are not aware of their situation (<3 months of gestation). These women were considered in the present study in terms of the cytogenetic damage that they might induce to their children through smoking. Moreover, to the authors’ knowledge, this is the first study to investigate the cytogenetic effect of pre-pregnancy smoking using the mutagenic agent CPT-11. Increasing frequency of SCEs indicates the existence of genotoxicity, and therefore increasing DNA damage in cells due
to several mutagenic factors [9,31]. The present results show that the DNA of newborns whose mothers smoked before pregnancy had chromosome instability, as shown by the increased SCE levels of umbilical cord blood lymphocytes exposed to CPT-11. This indicates that DNA damage was caused in the early stages of development, such as the embryonic stage. Obviously, it is not possible to control all cases of pregnancy, but women who are planning to have children should be advised to quit smoking well before conception in order to improve the health of their foetus. It has been known for many years that certain tobacco metabolites can cross the placenta and become toxic to the foetus [18,42–44]. It seems that smoking cessation in the early stages of pregnancy is not sufficient to avoid this. Apart from the direct adverse effects of smoking, there is also indirect damage such as cytogenetic damage, reflected by the increased frequency of SCEs. This type of damage can act synergistically with other environmental and/or chemical mutagenic substances to which people are exposed, willingly or otherwise, leading to carcinogenicity [13]. Observations of daily life raise concern that tobacco, alcohol and drugs are increasingly pressing problems, including among young/ pregnant women. This is both a medical and a socio-economic issue. In conclusion, the data indicate a strong association between maternal smoking before pregnancy and their newborns. The DNA of newborns whose mothers smoked before pregnancy appeared to have chromosome instability, shown by higher levels of SCEs in umbilical cord blood lymphocytes exposed to CPT-11. However, the increased proliferation capabilities and high survival potency of umbilical cord blood lymphocytes in control cultures (not exposed to CPT-11) suggest that these cells are dynamic. The genotoxic effects of smoking can be considered as important risk factors for cancer and/or genetic-related diseases. It appears that smoking cessation in early pregnancy is not sufficient, so it is strongly recommended that smoking cessation programmes should re-evaluate existing results and consider new directions for women planning to have children. The authors believe that smoking cessation needs to occur well before conception in order
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