Mutagenesis Advance Access originally published online on February 6, 2007
Mutagenesis 2007 22(2):123-127; doi:10.1093/mutage/gel062
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In vitro susceptibilities in lymphocytes from mothers and cord blood to the monofunctional alkylating agent EMS
School of Life Sciences, University of Bradford, Bradford, UK 1Maternity Department 2Department of Diabetes and Endocrinology, Bradford Royal Infirmary, UK 3Center for Health and Society, University of Copenhagen, Copenhagen, Denmark
It has been reported that children may experience different levels of chemical exposures than adults and that their sensitivities to chemical toxins may be increased or decreased when compared to adults. The perinatal period is one period in which these susceptibilities may be examined. Midwives at the Bradford Royal Infirmary collected venous blood samples from mothers at the time of birth and venous cord blood post-delivery. Lymphocytes were isolated from both blood types and examined in the alkaline comet assay using the monofunctional alkylating agent ethyl methanesulphonate (EMS). There were no biologically significant differences when subjects were categorized into subgroups based on lifestyle habits and physical characteristics, and overall there were no statistically significant differences in levels of DNA damage in mothers (n = 22) and babies (n = 22), except at the basal level (P < 0.05), but mean values in babies were always lower over the EMS dose range. Whole blood was used in the micronucleus (MN) assay, and there was a significantly (P < 0.05) higher rate of MN in mothers (n = 17), per 1000 binucleates, as compared with lymphocytes from their offspring (n = 17) at the basal level. This may be accounted for by age and endogenous factors. Overall, this current study cannot provide statistically significant evidence that children have either increased or decreased levels of susceptibility to a chemical toxin in comparison to adults when EMS is examined in vitro.
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Introduction |
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A number of health provisions have arisen because of the impact that certain environmental chemical exposures may have upon the health of the young (1
). It was reported that children may experience different levels of chemical exposures than adults and that their sensitivities to chemical toxins may be increased or decreased in comparison to adults (1). These considerations also apply to the unborn and the newborn (2).
The perinatal period includes the time shortly before and post-birth. Many biochemical and physiological changes occur in the foetus/child at this point (2). The placenta itself becomes fully formed during the early weeks of placental life, allows transport of molecules and synthesizes and metabolizes a number of protein and steroid hormones (3). It may also play an important role in protecting the unborn child from infections or antigenic challenges (4). The newborn has almost normal levels of T cells, T-cell subsets and a proliferative response to mitogens and allogeneic cells, but specialized immune functions are still inadequate at this point. Respiration also differs within the newborn. Foetal blood has a high oxygen affinity, making it well-adapted for placental transfer, and postnatal changes cause a rapid increase in oxygen affinity and capacity for oxygen carrying (5). The gaseous composition of the placenta and umbilical cord at the time of birth may be varied depending on the quality of the foetal oxygen supply, and it may be that a Caesarean section affects the gaseous composition (5). Newborn metabolism also includes the advent of glycogenolysis, an increase in free fatty acids, a number of endocrinal changes and the advent of continuous breathing (2).
The ability of newborns and young children to detoxify and excrete chemicals is dependent upon the maturity status of enzyme transformation systems (2); most hepatic enzymes do not reach adult levels until post-partum (6). The P450 enzymes develop at various rates during gestation, and usually at the time of birth are approximately one-third the adult rate (7). Because P450 enzymes are involved in the metabolism of lipid-soluble chemicals (8), the gestational transformation of chemicals (toxic or otherwise) may be hindered by undeveloped cytochromes (2). The role of maternal exposures on the foetus also remains unelucidated, although it has been shown that lifestyle choices such as smoking and alcohol use may affect the frequency of translocations in the foetus (9). It was found (10) that studies which examine environmental pollutant exposure in children show higher levels of micronuclei (MN) and sister chromatid exchanges (SCEs), as well as higher levels of DNA damage as observed in the comet assay (10).
The European Network on Children's Susceptibility and Exposure to Environmental Genotoxicants (CHILDRENGENONETWORK) was developed in 2001. Its role (10,11) was to collect information from participating laboratories and also to review literature in order to evaluate the strength of association between genotoxic compounds and the period of early life. The network also initiated pilot studies with the aim of comparing susceptibility of adults and children. This present study is one of these nationally part-supported studies.
Ethyl methanesulphonate (EMS) is a monofunctional alkylating agent, which donates a single ethyl group to reactive nucleophilic sites within DNA (12). Its toxicity and mutagenicity have been observed in a number of toxicological studies (13,14), and is therefore a valid compound to use in studies of this current nature because of its known effects in vitro. It has also been used as a model compound for sometime (15). EMS is predominantly a point mutagen and induces a high level of these point mutations through the formation of O6-ethylguanine and consequent mispairing to give G to A transitions (16). EMS is also able to induce SCEs (12).
This present study used EMS to examine differences to in vitro DNA damage in lymphocytes from mothers and from cord blood lymphocytes of their offspring to determine if there were differences in levels of susceptibility between the two groups.
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Materials and methods |
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Participant data collection
The appropriate midwife/birthing partner completed the mothers' questionnaire. Information obtained included age, occupation, health status, medication, as well as questions concerning pregnancy history. A second questionnaire was then completed on behalf of the baby. Sex and birth weight (g) were determined. Gestation period and delivery mode were recorded, alongside other physiological data.
Ethics and sample collection
Venous blood samples were taken from volunteers attending the Maternity Department at the Bradford Royal Infirmary. Inclusion criteria were based on willingness to participate in the study. Informed consent was obtained from all subjects and the study was approved by the local Research Ethics Committees (Bradford National Health Service Trust). Subjects were allowed to withdraw from the study at any time, and the samples were obtained over a period of 18 months. Approximately 30 ml of blood was taken from mothers by midwives via standard venipuncture using lithium-heparin sampling tubes (Sarstedt Ltd, Leicester, UK) as near to delivery time as possible. The midwife collected venous cord blood into a separate lithium-heparin sampling tube at an appropriate time post-delivery. All babies were in good condition at birth and needed no resuscitation. Whole blood was used in the MN assay, and lymphocytes were isolated using standard separation medium (Lymphoprep©, Axis shield, Oslo, Norway) and immediately frozen stepwise in foetal calf serum (FCS) [10% dimethyl sulphoxide (DMSO)] and transferred to long-term liquid nitrogen storage. All frozen samples were checked at the time of assay for viability post-thawing using trypan blue exclusion.
Chemicals
The chemicals used in these experiments were purchased from the following suppliers: low and normal melting point agarose, L-glutamine, penicillin/streptomycin, RPMI w/o phenol red, phytohaemagglutinin-M (PHA-M) from Gibco Invitrogen, Paisley, UK. Cytochalasin B [CAS-14930-96-2], DMSO [CAS-67-68-5], ethidium bromide (EtBr) [CAS-1239-45-8], EMS [CAS-62-50-0], ethylenediaminetetraacetic acid [CAS-6381-92-6], formaldehyde [CAS-75-12-7], mitomycin C [CAS-50-07-7], Triton X-100 [CAS-9002-93-1] and 0.4% trypan blue [CAS-72-57-1] from Sigma Chemicals Co. Ltd, Poole, Dorset, UK. Ethanol (EtOH) [CAS-64-17-5], Giemsa Gurr, glacial acetic acid [CAS-64-19-7], methanol [CAS-67-56-1], potassium chloride (KCl) [CAS-7447-40-7], potassium phosphate monobasic [CAS-7778-77-0], disodium hydrogen phosphate dehydrate [CAS-10028-24-7], sodium hydroxide [CAS-1310-732] from BDHMerck, Poole, Dorset, UK. Lymphoprep© from Axis shield, Oslo, Norway. FCS from SeraQ, Sussex, UK. Phosphate-buffered saline from Oxoid, Hampshire, UK. Histomount from Raymond A Lamb, Sussex, UK.
The comet assay
The alkaline comet assay was used as previously described (17–19). Lymphocytes were quickly thawed and suspended in RPMI, with or without EMS. Cells were incubated at 37°C for 30 min. The dose range of EMS was determined when the viability of lymphocytes at all doses was >90% using trypan blue exclusion (20). Lysing time was between 1 and 3 h. Unwinding was 30 min, with electrophoresis for 30 min (25 V, 
300 mA). Coded slides were stained with EtBr post-neutralization and viewed (x20 objective, x10 lens) using a fluorescent microscope (Leica, Wetzler, Germany) equipped with appropriate filters. A charge-coupled device camera connected the microscope to an image analysis system (Kinetic Imaging, Liverpool, UK) incorporating the analysis software Komet 4. The parameter for analysis was tail moment (tail length multiplied by intensity). At least 50 cells were scored from replicate slides per dose per subject.
The MN assay
To each culture tube, whole blood of 500 µl was added [82% RPMI, 15% FCS, 1% PHA-M (1 mg/ml), 1% L-glutamine, 1% penicillin/streptomycin], making a total volume of 5 ml. Mitomycin C (0.01 µM) was used as a positive control. Cultures were incubated at 37°C for 72 h. Required treatment (max 1% of culture volume) was added 24 h after the start of incubation. The levels of EMS used were optimal values attained through multiple experiments, and corresponded to relevant guidelines (21). At 44 h, 10 µl of cytochalasin B was added, and at 72 h, fixation was carried out. Centrifugation was for 8 min (120x g). The supernatant was removed via aspiration and the pellet resuspended. KCl was added up to a total volume of 5 ml while vortexing the tube. The second centrifugation was at 120x g for 8 min, and the supernatant was removed via aspiration and resuspension of the pellet. Carnoy (1 : 3 glacial acetic acid : methanol) was slowly added (up to 5 ml) while vortexing, and then a small quantity of formaldehyde was added to each tube. Centrifugation was as above, and the Carnoy wash and fixing were repeated twice. After removal of the supernatant, 20 µl of cell suspension was dropped in two spots, on EtOH-cleaned slides. Density was checked under an inverted microscope (Leica, Wetzler). Slides were covered with filter paper and left to dry at room temperature. Staining was carried out for 10 min in a premixed Giemsa Gurr staining solution (1 : 9 Giemsa : phosphate buffer), then rinsed in H2O. Slides were left to dry then cover slips mounted. Slides were scored as per human micronucleus guidelines (21) under x100 oil immersion objective (Leica, Wetzler).
Statistics
Data obtained from the comet assay violated the normality assumption, so non-parametrical data analysis (Mann–Whitney) was used to test between group (i.e. individual covariates) differences. A general linear model was also applied to test further between subject effects. MN data obtained confirmed the test for normality; therefore, the Student's t-test was used to assess differences between the population groups. The statistical software SPSS (version 11) was used to carry out all analyses and the graph was plotted in SigmaPlot (version 8). Despite statistically significant differences, values were only considered biologically significant if there were consistent dose-related increases with no overlapping of curves of mother and baby.
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Results |
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Comet assay
Table I demonstrates the effect of EMS on DNA damage in the comet assay in lymphocytes from mothers and babies, and subgroups of mothers and babies, as measured by the mean tail moment ± standard error of the mean. No statistically significant differences in levels of DNA damage were observed between mothers and babies, except at the basal level (P < 0.05). However, the mean values of damage always appeared to be higher for mothers than for babies, but the trend was not statistically significantly different, and error bars did overlap in a number of doses.
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DNA damage in mothers categorized by smoking status is shown, and there were no statistically significant differences in levels of damage, except at the 1-mM level (P < 0.01). Significantly more DNA damage (P < 0.05) was observed in lymphocytes at the 1-mM level from non-/former alcohol drinkers and at the 10- and 20-mM level (P < 0.01). No statistically significant differences in induced DNA damage were observed between mothers who ate a Western-type diet and lymphocytes from mothers who ate an Asian-type diet, except at the 40-mM level (P < 0.05). There was no difference in levels of damage between lymphocytes from mothers with one or more pregnancies.
No statistically significant differences in DNA damage were observed between lymphocytes from male and female offspring. There was significantly higher damage at the 1-mM level (P < 0.05) in cord blood lymphocytes from offspring whose mothers were non-/former smokers. When results from cord blood lymphocytes were categorized as per mothers' diet type, there was significantly more damage seen at the 20-mM level (P < 0.05) in cord blood lymphocytes from mothers who ate an Asian-type diet, and there was significantly more damage (P < 0.05) at the 1-, 10- and 20-mM level in lymphocytes from those offspring born via Caesarean section. Again there was no evidence of a linear trend.
The MN assay
Figure 1 shows the rate of MN per 1000 binucleates (BN) in lymphocytes from mothers and from cord blood of their offspring. There were significantly (P < 0.05) more MN observed in mothers compared with their offspring at the basal level; there were no significant differences in numbers of MN when EMS was introduced into the assay.
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Discussion |
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Over the last few years, research into differences in genetic susceptibility between mothers and their offspring has begun to increase, as it is becoming apparent that there are considerable differences between the adults and the young in the detoxification and excretion of chemicals (2
,22).
To an extent, this has been understood for sometime with a large number of experiments both in vivo and in vitro that have examined many different chemicals and compounds to see to what extent genetic damage is continued through germ lines (23). It is also known that a number of maternal exposures, including lifestyle choices such as smoking and alcohol use and abuse, during gestation increase the risk of teratogenic effects upon the foetus, often resulting in either the miscarriage or the birth of an offspring with abnormalities (24). The perinatal period, the time shortly before and after birth, is one of the areas in which there appears to be a lack of research. Some of the reasons may be that it is hard to distinguish the role of endogenous effects upon the foetus, such as maternal hormonal imbalances, and other physical changes arising through birth preparation. It may also be difficult to assess such factors as smoking on the foetus around the time of birth [although the teratogenic effects of smoking during gestation are well-known (25–27)], and SCE frequencies in smoking mothers are consistently higher than the levels of SCEs in the offspring (10). Other factors may include physiological well-being of the foetus before and at the time of birth, in which disorders such as pre-eclampsia may affect the physical well-being of both mother and offspring.
It also should be considered that infants are not a homogeneous population, since significant differences are observed in the results of many neonatal tests (28) and that it is difficult to define normality in this particular human population. It also has been reported that test ranges defined for children are inappropriate when testing newborns or premature infants (28).
There were no biologically significant differences between smoking and non-smoking mothers, although the low number of smokers volunteering for the study has prevented accurate statistical analyses from being produced. These findings cannot support previous studies (10), which showed that biomarkers are consistently increased in those who are transplacentally exposed to either smoking mothers or environmental tobacco smoke (10). There were no biologically significant differences between mothers who do or do not drink alcohol, and in relation to diet, mothers whose dietary intake consists of the Asian-type diet did not sustain any differences in induced levels of DNA damage compared with those eating the Western-type diet. This same result is mirrored in differences between DNA damage in the cord blood samples, where results were segregated depending upon their mothers' dietary intake, and the lymphocytes from babies whose mothers' diet was Asian type sustained higher DNA damage at one level, but the lack of further statistical differences prevents extrapolation at this point. We have also shown that there were no significant differences between lymphocytes from those whose current pregnancy was the first and lymphocytes from those who have had previous pregnancies, neither can our current study provide support for previous work, which has shown differences in DNA damage between males and females (29–31).
This study has shown some differences in induced DNA damage occurring between lymphocytes from babies who are delivered by a Caesarean section and those who are delivered via a normal vertex (vaginal delivery). A Caesarean section may be planned for a number of reasons, including maternal wishes, complications or previous sections, or an emergency Caesarean section may also be carried out if the birth fails to progress.
There was a significantly higher level of MN per 1000 BN in lymphocytes from mothers compared with babies. This confirms previous studies (32,33), one of which (32) showed that mothers had a higher level of MN at the basal level, even those residing in different geographical areas (although some of the mothers surveyed were exposed to pesticides during pregnancy). It is also reported (32) that the influence of medication on their results was not correlated. Previous studies have also concluded a relationship between age and an increase in levels of MN (34,35), or other factors such as smoking (30) and work exposures (36) may contribute to higher levels of MN observed in individuals.
However, it should be noted that the low numbers of subjects examined impact upon earlier statements of significance levels, and although the tests used are appropriate for lower subject numbers, a generalization to larger populations cannot be made at this time, although homogeneity indicates reliability of the obtained data.
Concerning the in vitro treatment with EMS, the action of the compound appeared to be similar in different population groups. Although it should be noted that there were major differences in optimal levels of EMS, in relation to the length of treatment time in both assays, a short-term treatment in the comet assay allowed the use of much higher dosage, and the effects of the compound are measured in non-dividing cells, whereas the 20-h treatment time and the use of proliferating cells in the MN assay required a lower treatment level. There were also no differences between the cytochalasin B proliferation index in the two groups (data not shown), which is an indicator of the number of cell cycles per cell during the period of exposure to cytochalasin B, showing that the rate of lymphocyte cycling between mothers and babies was almost identical.
It is known that effects are exerted upon mammalian cells in vitro after treatment with EMS (37), and the influence of genotypic traits upon individual susceptibilities requires consideration in most types of toxicity exposure (38). However, because EMS is a direct acting alkylating agent, not requiring metabolic activation, the impact that genotypes may otherwise have upon the absorption, distribution, metabolism and excretion of the compound is reduced considerably, especially with the use of in vitro studies such as in the present study.
Overall, it appears that there were no differences in levels of in vitro-induced DNA susceptibilities between lymphocytes from mothers and babies using the compound EMS in the comet and MN assays. There was a significant difference at the basal level for the incidence of MN, but this present study cannot provide evidence that children have either increased or decreased susceptibility/sensitivity (2) to the monofunctional alkylating agent EMS in comparison to adults when examined in these two in vitro assays.
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Acknowledgments |
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We thank all patients and staff from the Bradford Royal Infirmary for their support in the conduct of this study. The study was part funded by Grant QLK4-CT-2002-02198 from the European Network on Children's Susceptibility and Exposure to Environmental Genotoxicants (CHILDRENGENONETWORK).
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* To whom correspondence should be addressed. Tel: +44 1274 233579; Fax: +44 1274 309742; Email: d.anderson1{at}bradford.ac.uk
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References |
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Received on July 3, 2006; revised on October 25, 2006; accepted on November 9, 2006.
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