Mutagenesis Advance Access originally published online on April 7, 2005
Mutagenesis 2005 20(3):181-185; doi:10.1093/mutage/gei022
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Interaction between cadmium and aromatic DNA adducts in hprt mutagenesis during foetal development
Department of Health Risk Analysis and Toxicology, University of Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands, 1Department of Obstetrics and Gynecology, University Hospital Maastricht (AzM), PO Box 5800, 6202 AZ Maastricht, The Netherlands and 2Research Institute Growth and Development (GROW), University of Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands
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Abstract |
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The foetus is exposed to multiple xenobiotics through the mother's circulation and this is possibly involved in the development of diseases in later life. Heavy metals and lipophilic genotoxins in umbilical cord blood of newborns may have synergistic effects on mutagenesis in the hypoxanthine-phosphoribosyltransferase (HPRT) reporter gene. Concentrations of zinc (Zn), lead (Pb) and cadmium (Cd) were determined in the peripheral and cord blood of 16 non-smoking and 9 smoking healthy mothers by atomic absorption spectrometry. Lipophilic DNA adducts in lymphocytes were determined in the same subjects by 32P-postlabelling and the HPRT-variant frequency was assessed by the evaluation of 6-thioguanine resistant cells. Although the Cd/Zn ratio was 2.5-fold higher in the blood of smoking women than in non-smoking women (1.0 ± 0.2 and 0.4 ± 0.1, respectively, P = 0.007), this difference could not be observed in umbilical cord blood (0.3 ± 0.1 and 0.3 ± 0.1, respectively, P = 0.66). Similarly, mean DNA adduct levels were increased in the lymphocytes of smoking women compared with non-smoking controls (0.99 ± 0.31 adducts/108 nt and 0.43 ± 0.12, respectively, P = 0.009), but were only marginally higher in the neonates of smokers than in their non-smoking counterparts (0.57 ± 0.29 and 0.24 ± 0.09, respectively, P = 0.38). Since Cd is known to effectively inhibit DNA repair, we hypothesized that concomitant exposure of neonates to Cd and genotoxic compounds may result in an increased fixation of DNA damage into somatic mutations. Indeed, the number of HPRT-variants per adduct (i.e. the mutagenic efficiency of adducts) correlated positively with the Cd concentrations in cord blood (r = 0.61, P = 0.001). These data suggest a molecular link between DNA damage, inhibition of DNA repair by Cd and in vivo mutagenesis during foetal development. Thus, exposure to heavy metals may enhance the mutagenic potential of DNA-damaging compounds and results in biologically relevant genotoxic effects in neonates.
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Introduction |
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It is well known that the foetus is exposed through the placenta to DNA-damaging xenobiotics, which are present in the peripheral circulation of the mother. Indeed, tobacco smoke related compounds have been detected in the umbilical cord blood, placenta and urine of neonates of smoking mothers (1
–4). Important genotoxins in cigarette smoke are polycyclic aromatic hydrocarbons (PAH). PAH are metabolized to reactive derivatives, which can covalently bind to DNA (i.e. DNA adducts), and are thought to be involved in the onset of carcinogenesis (5). Although foetal exposure to such carcinogenic agents is evident, the role of maternal smoking during pregnancy in childhood cancer was found to be small for neonates when considered as a whole (6). However, these exposures may become more relevant in newborns that are simultaneously exposed to co-carcinogens like toxic heavy metals [e.g. lead (Pb) and cadmium (Cd)]. Even though Cd is carcinogenic, its mutagenic potential is rather weak. In contrast, Cd exerts pronounced co-mutagenic effects, which may be explained by its inhibition of nucleotide excision repair processes (7). Although the placenta may function as a barrier for heavy metals between mother and child, preventing increased concentrations of these toxic elements in the foetus, these metals are still detectable in umbilical cord blood (4,8). Therefore, it can be hypothesized that the induction of mutations by carcinogen–DNA modifications is more predominant in those neonates with a concomitant exposure to heavy metals.
DNA damage recognition by specific proteins is required to effectively initiate DNA repair. After an addition of Cd, a dose-dependent reduction of such protein–DNA interactions was observed in vitro, which could be restored by the addition of zinc (Zn) (7). Thus, PAH–DNA lesions could persist at relatively high Cd/Zn ratios, because of ineffective DNA repair. DNA damage that is not repaired or not efficiently repaired may result in the misincorporation of a nucleotide opposite the damaged site, particularly in rapidly dividing foetal cells. As a consequence, the nucleotide sequence is altered, which is thought to be a critical initial step in carcinogenesis, because mutations in cell-cycle regulatory genes may disrupt normal cell growth. Mutations in the gene for hypoxanthine-phosphoribosyltransferase (HPRT) are easily detectable, but are not necessarily involved in the process of carcinogenesis. However, HPRT-mutations are considered to reflect mutational events in genes relevant for cell-cycle control (9) and can be detected in the cord blood from neonates (10).
Thus, even at low levels of exposure to both DNA damaging-compounds and heavy metals, the combination of both may still result in significant mutagenic effects. Simultaneous in utero exposure of neonates to genotoxins and heavy metals interact in mutagenesis during foetal development. For this purpose, aromatic DNA adducts and heavy metals were assessed in umbilical cord blood and were linked to the number of mutations in the HPRT-reporter gene. It should be mentioned here that it was not the purpose of this work to thoroughly investigate the effect of smoking on foetal mutagenesis. Smokers and non-smokers were rather selected to create a broad exposure-range to PAH as well as heavy metals.
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Materials and methods |
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Study population
Twenty-five first-time pregnant healthy women from the area of Maastricht and Heerlen in the Netherlands were selected. Smoking mothers (n = 9, mean age ± SD: 31 ± 3 years) reported to smoke 9 ± 3 cigarettes/day and continued to smoke until a few hours before delivery. Non-smoking women (n = 16) were 31 ± 2 years of age. Pregnancies and deliveries were without complications or prescription of medication. All the women answered a questionnaire regarding their potential exposure to genotoxic compounds and smoking behaviour. This study was approved by the medical ethical committee of the Maastricht University.
Isolation of blood cells
Ten millilitres of umbilical cord blood (child) and peripheral blood (mother) were obtained by venapuncture into EDTA-tubes, stored at 4°C, and processed within 18 h. Mononuclear cells were isolated by centrifugation on LymphoprepTM (1.077 g/ml, Nycomed, Oslo, Norway) according to Bøyum (11). The mononuclear cell fraction was washed twice with PBS and stored at –20°C until DNA isolation.
Analysis of Cd, Pb and Zn in maternal and cord blood by atomic absorption spectrometry
One volume blood (ca. 100 µl) was hydrolyzed with 3 vol of 1 M nitric acid (HNO3) for 30 min. After centrifugation, the supernatant was directly injected into a graphite furnace atomic absorption spectrometer with Zeeman background correction (Varian, Bergen op Zoom, The Netherlands). Cd, Pb (µg/l) and Zn concentrations (mg/l) were determined at 228.8, 283.3 and 213.9 nm, respectively. Solutions with known concentrations of Cd, Pb and Zn were used for calibration. All the glassware were rinsed with 1% HNO3 to avoid contamination.
DNA isolation and 32P-postlabelling
DNA was isolated using phenol extraction and 32P-postlabelling analysis was performed to assess DNA adduct levels (12). Briefly, DNA was enzymatically digested and unmodified nucleotides were dephosphorylated by nuclease P1. Labelling was carried out with excess [
-32P]ATP and T4-polynucleotide kinase. 5'-Labelled adducts were resolved on polyethylenimine–cellulose TLC sheets (Merck, Darmstadt, Germany), using the solvents as described by Godschalk et al. (12). In each experiment, three standards of [3H]benzo[a]pyrenediolepoxide modified DNA with known modification levels (1/107, 108 and 109 nt) were run in parallel for quantification purposes. Quantification was performed by using a phosphorimager (Molecular DynamicsTM, Sunnyvale, CA).
Assessment of the HPRT-variant frequency
The HPRT variant frequency (Vf) was determined by evaluation of 6-thioguanine resistant HPRT-variants. HPRT-variant lymphocytes were identified by immunocytochemical staining with monoclonal anti-bromodeoxyuridine (BrdU) (13). Briefly, cultured lymphocytes were treated with 6-thioguanine and after 24 h, cells were labelled with BrdU for 16 h. Cells were hypotonized, fixed and transferred to slides. These slides were screened for brown BrdU-containing cells by one well-trained observer.
Statistical analysis
Data are presented as mean ± SE. Relationships between various parameters were determined by both simple as well as multiple regression analysis. Non-parametric tests were applied to evaluate statistical significance, considering P< 0.05 as statistically significant.
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Results |
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Heavy metals in maternal and cord blood
The overall characteristics and results are presented in Table I. In maternal blood, Cd was the only metal of which the levels were significantly affected by the mother's smoking behavior (mean levels were 0.69 ± 0.14 and 0.33 ± 0.07 µg/l for smokers and non-smokers respectively, P = 0.04). Cd concentrations in their neonates were, however, not affected (0.07 ± 0.02 and 0.06 ± 0.01 µg/l for smokers and non-smokers respectively, P = 0.64). Zn or Pb concentrations were not different between smokers and non-smokers in maternal as well as umbilical cord blood (see Table I). As a result, the Cd/Zn ratio was increased in the peripheral blood of smoking mothers (1.0 ± 0.2 and 0.4 ± 0.1, respectively, P = 0.007), but not in their children (0.3 ± 0.1 and 0.3 ± 0.1, respectively, P = 0.66). Pb concentrations in cord blood correlated significantly with Pb concentrations in the mother (R = 0.89, P < 0.001). No such correlations were observed for Cd (R = 0.33, P = 0.13) or Zn (R = 0.10, P = 0.67). Overall, concentrations of all metals were significantly higher in mothers than in their neonates for both smokers as well as nonsmokers (Table I).
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Aromatic DNA adducts in mother and child
Aromatic DNA adducts were detected in smoking mothers as a diagonal radioactive zone (DRZ) by thin layer chromatography, which is typical for exposure to complex mixtures (Figure 1). In the neonates, DNA adducts were also detectable as a faint DRZ, but only if the mothers smoked throughout pregnancy. A single adduct spot was detected (Figure 1, panel E) in one sample. Aromatic DNA adducts were found to be higher in the peripheral blood of smoking mothers (0.99 ± 0.31 adducts/108 nt) than in non-smokers (0.43 ± 0.12, P = 0.009). Although a 2-fold difference was observed in umbilical cord blood (0.57 ± 0.29 and 0.24 ± 0.09, for neonatal blood of smoking and non-smoking mothers, respectively), this difference did not reach statistical significance (P = 0.38). Overall, DNA adduct levels were higher in mothers than in their neonates (P = 0.011) and a positive correlation between both was observed (R = 0.63, P = 0.001).
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HPRT-Vf and its correlation with heavy metals and DNA adducts
The overall HPRT-Vf was 3.9 ± 0.7/106 lymphocytes and ranged from 0.4 to 12.2. The HPRT-Vf appeared to be 3-fold higher in the cord blood of neonates from smokers (6.6 ± 1.4/106 lymphocytes, range: 0.4–12.2) than in the blood of neonates with non-smoking mothers (2.3 ± 0.4/106, range: 0.4–5.2, P = 0.03, Table I). A weak non-significant positive correlation was observed between the HPRT-Vf and DNA adduct levels (R = 0.31, P = 0.16). No significant correlation was observed between the HPRT-Vf and concentrations of Pb, Cd or Zn in umbilical cord blood. Multivariate analysis did not reveal any additional statistically significant relationships.
To estimate the efficiency by which DNA adducts are converted into mutations, the HPRT-Vf per adduct ratio was calculated, and a good correlation with the Cd (R = 0.61, P = 0.001) as well as the Cd/Zn status was observed (R = 0.57, P = 0.002) (Figure 2). The calculated HPRT-Vf per adduct ratio was independent from maternal smoking behaviour and was not affected by Pb or Zn concentrations in umbilical cord blood.
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Discussion |
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Epidemiological studies showed that the overall childhood cancer risk as a result of maternal smoking is relatively small (6
), but still, some individuals may be at a significantly increased risk owing to synergistic effects of co-exposures to toxic chemicals. For instance, cigarette smoke derived carcinogens bind covalently to DNA to form pro-mutagenic DNA adducts, which can be permanently converted into mutations if not removed by DNA repair processes at the appropriate time. Cell proliferation is high in unborn children, thus, removal of DNA damage before DNA doubling in S-phase to prevent fixation of DNA damage into a mutation, is of high importance. Cigarette smoke contains high levels of heavy metals, which compete with the essential element Zn. As a result, nuclear enzymes that rely on zinc-fingers, like DNA repair enzymes of the nucleotide excision repair pathway, can lose their functionality (7,14). Thus, in neonates with relatively low DNA adduct levels, the conversion of pro-mutagenic DNA lesions into mutations may occur more efficiently if there is co-exposure to heavy metals. This hypothesis was tested by the simultaneous assessment of DNA adduct levels and the presence of heavy metals (Cd and Pb) in neonates from a selected group of non-smoking mothers and their relation with the in vivo formation of phenotypic variants in the HPRT-reporter gene.
In the present study, Zn concentrations were unaffected in maternal and foetal blood by maternal smoking. On the contrary, Cd concentrations were increased in the maternal blood of smokers compared with non-smokers, but not in umbilical cord blood. Similar results were obtained by others (4,15). This can be explained by the induction of metallothioneins in placenta to block the transfer of Cd from the mother to the child (16,17). Although, the placenta seems to be a good (but not perfect) barrier for Cd, Pb crossed the placental barrier more easily as demonstrated by a strong correlation between maternal and umbilical Pb concentrations. Nevertheless, Pb levels were significantly lower in the neonates compared with their mothers. These data are in line with previous work in which the placental passage of Pb is well described (4,8). In the present study, maternal and foetal concentrations of Zn, Cd and Pb were in line with previously published levels (4).
DNA adduct levels were higher in the neonates of smoking mothers than in the newborns of non-smoking mothers, verifying that tobacco smoke derived carcinogens or their metabolites, pass the placenta and reach the foetus (1,18). However, this difference between DNA adduct levels in the neonates of smokers and non-smokers did not reach statistical significance, which is in line with Whyatt et al. (19) who also showed that maternal smoking (active and passive) significantly increased maternal, but not newborn DNA adduct levels. Interestingly, in the present study, DNA adduct levels were lower in children than in their mothers, which is in contrast to the results by Whyatt et al. (20) and Perera et al. (21). In a recent prospective study, increased DNA adduct levels were related to higher cancer risks in adults (5). Foetal tissues are believed to be even more sensitive to DNA damage, because of the high proliferation rate, which is a prerequisite for the fixation of DNA damage as a mutation. Indeed, the HPRT-Vf in newborns of smoking mothers was 3-fold increased. Although mutations in the HPRT-gene are not necessarily involved in carcinogenesis, they are considered to report mutation frequencies in cancer-prone genes. The literature on the induction of HPRT-mutations by maternal smoking shows conflicting results. The increased levels of HPRT-variants in cord blood may originate (at least partly) from the promu-tagenic lesions formed by aromatic genotoxic compounds from cigarette smoke, which is also suggested by others (10,20). On the other hand, others were unable to find an effect of cigarette smoking on HPRT-mutagenesis using the T-cell cloning assay (22,23). This intriguing discrepancy between the results obtained by different methodologies deserves further attention before firm conclusions about the induction of HPRT mutations in cord blood by maternal smoking can be made.
In the present study, the efficiency with which DNA lesions were converted into mutations was estimated by calculation of the number of variants per adduct. This ratio was strongly related with the Cd status of the foetus, indicating that Cd potentiates the fixation of DNA damage, possibly as a consequence of DNA repair inhibition. However, this interpretation of the data still needs some caution, because DNA repair capacity was not measured directly and an increased mutation rate at low aromatic DNA adduct levels could also indicate that other pro-mutagenic lesions are involved, for example, DNA lesions induced by Cd-related oxidative stress (24). On the other hand, Pb exposure may also result in oxidative stress and in the present study no effect of Pb on the mutagenic efficiency of adducts was observed. This is in line with in vitro experiments showing that Pb was ineffective in inhibiting nucleotide excision repair enzymes (25).
Overall, these data indicate that genetic damage is converted into mutations more efficiently if there is co-exposure to relatively high levels of Cd in the developing child. Thus, there is a profound concern for the possibility of carcinogenic effects in newborns of smoking mothers. Future research on foetal carcinogen exposure and elimination, should therefore consider inter-individual differences in the activity of DNA repair enzymes and concomitant exposure to heavy metals of both mother as well as the foetus.
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* To whom correspondence should be addressed. Tel: +31 433881104; Fax: +31 433884146; Email: R.Godschalk{at}GRAT.unimaas.nl
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