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Maternal hemochromatosis gene H63D single-nucleotide polymorphism and lead levels of placental tissue, maternal and umbilical cord blood
Environmental Research 140 (2015) 456–461
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Maternal hemochromatosis gene H63D single-nucleotide polymorphism and lead levels of placental tissue, maternal and umbilical cord blood Zeliha Kayaalti a,n, Dilek Kaya-Akyüzlü a, Esma Söylemez a,b, Tülin Söylemezoğlu a a b
Ankara University, Institute of Forensic Sciences, Ankara, Turkey Middle Black Sea Passage Generation of Agricultural Research Station Director, Tokat, Turkey
art ic l e i nf o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 30 April 2015 Accepted 5 May 2015
Human hemochromatosis protein (HFE), a major histocompatibility complex class I-like integral membrane protein, participates in the down regulation of intestinal iron absorption by binding to transferrin receptor (TR). HFE competes with transferrin-bound iron for the TR and thus reduces uptake of iron into cells. On the other hand, a lack of HFE increases the intestinal absorption of iron similarly to iron deficiency associated with increasing in absorption and deposition of lead. During pregnancy, placenta cannot prevent transfer lead to the fetus; even low-level lead poisoning causes neurodevelopmental toxicity in children. The aim of this study was to determine the association between the maternal HFE H63D single-nucleotide polymorphism and lead levels in placental tissue, maternal blood and umbilical cord bloods. The study population comprised 93 mother–placenta pairs. Venous blood from mother was collected to investigate lead levels and HFE polymorphism that was detected by standard PCR–RFLP technique. Cord bloods and placentas were collected for lead levels which were analyzed by dual atomic absorption spectrometer system. The HFE H63D genotype frequencies of mothers were found as 75.3% homozygote typical (HH), 23.6% heterozygote (HD) and 1.1% homozygote atypical (DD). Our study results showed that the placental tissue, umbilical cord and maternal blood lead levels of mothers with HDþ DD genotypes were significantly higher than those with HH genotype (po 0.05). The present study indicated for the first time that mothers with H63D gene variants have higher lead levels of their newborn's placentas and umbilical cord bloods. & 2015 Elsevier Inc. All rights reserved.
Keywords: Human hemochromatosis protein Single nucleotide polymorphism Maternal blood Umbilical cord blood Placental tissue Lead
1. Introduction Iron is an essential and also vital trace element that is a component of many hemoproteins and non-heme iron proteins. It plays a critical role in oxygen sensing and transport, electron transfer, and catalysis. However, when present in excess and not tightly bound, it can be toxic by catalyzing the production of oxygen radicals. Thus, the levels of iron in the body are tightly controlled to prevent either iron depletion or iron overload by several proteins such as transferrin, divalent metal transporter-1 (DMT-1), ferroportin, hemochromatosis (HFE) and hepcidin (Horvathova et al., 2010; Cantonwine et al., 2010; Rolfs et al., 2002). The human HFE protein participating in the down regulation of iron absorption is a major histocompatibility complex class I-like integral membrane protein which is expressed in crypt cells of duodenum and syncytiotrophoblast cells of placenta (Rolfs et al., n
2002; Waheed et al., 1999; Akesson et al., 2000; Hanson et al., 2001). HFE does not bind or transport iron, but rather competes with iron-loaded transferrin for binding to the transferrin receptor (TfR) (Rolfs et al., 2002; Townsend and Drakesmith, 2002). HFE gene first identified as HLAH by Feder et al. in 1996 is located at 6p21.3 and covers approximately 10 kilobases and almost 4.6 megabases telomeric from HLA-A (Hanson et al., 2001). Among 37 allelic variants of this gene, the commonly found two variants C282Y and H63D (Hanson et al., 2001; Wang et al., 2007) are associated with hereditary hemochromatosis (HH) that is an autosomal recessive blood disorder characterized by an increase in iron absorption by comparison to iron excretion (Rolfs et al., 2002; Wang et al., 2007; Elmrghni et al., 2011). Elevated iron absorption is supposed to go along with increased absorption of chemically related divalent metals such as lead (Elsenhans et al., 2011). Although lead is a naturally occurring heavy metal, a local and
Correspondence to: Institute of Forensic Sciences, Ankara University, Dikimevi, 06590 Ankara, Turkey. Fax: þ90 312 3192077. E-mail address: [email protected] (Z. Kayaalti).
http://dx.doi.org/10.1016/j.envres.2015.05.004 0013-9351/& 2015 Elsevier Inc. All rights reserved.
Z. Kayaalti et al. / Environmental Research 140 (2015) 456–461
more diffuse lead has been enriched in the indoor and outdoor environments due to industrial development, urbanization and lifestyle factors and, thus, lead exposure becomes one of the most important health hazards (Bierkens et al., 2011). The main sources of lead contamination in soil and lead pollution in air are leaded gasoline and dust from lead-based house paints. Other sources are cigarette smoke, lead smelting industries, fuels used for heating, battery recycling plants and occupational exposure in automotive and aircraft paint manufacturing (Dorea, 2004). Lead, having no beneficial role to humans, can be received through food, contaminated air and dust, and accumulates particularly in the bones with a half-life of around 30 years (D’Souza et al., 2003; Tekin et al., 2012). Lead has adverse effects to mainly renal, hematopoietic and neurologic systems. The most susceptible populations to the adverse neurodevelopmental effects of lead are children, particularly infants in the neonatal period and fetus since neurodevelopment begins in this period and the blood–brain barrier is still immature (Goyer, 1990; Goyer, 1997; Molina et al., 2011). Moreover, exposure levels that do not harm the mother can be fetotoxic because of the differences between the many biochemical pathways of the adult and the fetus (Baghurst et al., 1991; Al-Saleh et al., 2011). Fetal exposure to lead begins at the 21st week of pregnancy and continues throughout the life (Goyer, 1990). Although the placenta, an interface between the developing fetus and the mother, has mechanisms that restrict the entry of toxicants (Al-Saleh et al., 2011), it cannot protect the fetus from exposure to lead from the mother's blood. In experimental models, it was demonstrated that umbilical cord blood flow rate affected the transfer of lead linearly (Goyer, 1990). Furthermore, iron deficiency or dietary iron consumption of the mother can also alter lead uptake (Molina et al., 2011). Hence, genes related to iron metabolism could modify lead absorption and pregnant women with genetic variants affecting iron metabolism could have children at increased risk of exposure to lead. To test this hypothesis, this study aimed to examine the effect of maternal HFE H63D single-nucleotide polymorphism (SNP) on mother–placenta–fetus lead levels. HFE gene was selected since (a) HFE influences the expression of other metal transporters such as DMT1 and ferroportin; (b) HFE is one of the genes that are recognized to influence lead toxicokinetics and toxicodynamics; and, (c) HFE protein is expressed at the apical membrane of syncytiotrophoblasts and placental iron transfer occurs at this site. Previously, Karwowski et al. (2014) studied the effect of maternal and/or infant hemochromatosis (both HFE C282Y and H63D) gene variants on maternal blood lead (MBL) and umbilical cord blood lead (UCBL). In this study, no statistically significant differences between geometric mean blood lead value and variant status was detected. Placental transfer of lead was found to be associated with only maternal HFE C282Y gene variant status, but not with maternal HFE H63D variants. Compared to Karwowski's study, we examined the effect of maternal HFE H63D (rs1799945; Gene ID:3077, Accession number: NP_001287678) gene variant status on lead levels of maternal and cord blood samples as well as placental tissues in Turkish population. Thus, to the best of our knowledge, this is the first study indicating that maternal HFE H63D gene variant status was significantly associated with lead levels of placental tissue and maternal and cord blood samples and may modify the placental transfer of lead in a triad of mother–placenta–fetus.
the Gynecology Department of Ankara University's Faculty of Medicine, from February to October in 2011. Mothers with gestational ages Z36 weeks (n ¼127) were screened in the hospital and asked to fill out a questionnaire, which included medical and dietary history, as well as data on occupational and potential environmental sources of metal exposure, and socioeconomic status. A small questionnaire used to gather demographic information was also given to individuals. Only Turkish subjects were included in the study. Healthy, non-smoking, non-anemic mothers (n ¼93) living in Ankara for more than 3 years without a history of alcohol, drug use or chronic disease were included in the study. Ineligibility criteria was a medical history of renal failure, diabetes, carcinoma, diagnosed hepatic or cardiovascular diseases that may be related to possible heavy metal accumulation from environmental or occupational exposures. Thirty-four women were excluded from the study due to occupational exposure (n ¼2, printing workers), smoking habit (n ¼25) and a medical history of chronic diseases (n ¼7). Written informed consent was obtained from those eligible for the study. Infant characteristics such as gestational age, birth weight, birth length and head circumference were also recorded (Table 1). Placenta, maternal and cord blood samples were collected at delivery by cesarean section or spontaneous labor. This study was approved by the institutional ethics committee (approval no: 33-730 in 2011). 2.2. Sample collection Two ml of venous blood was taken from each woman before delivery and from the umbilical cord during delivery into tubes with EDTA and stored at 4 °C. Placentas were collected and placed in a plastic bag immediately after delivery to avoid external metal Table 1 Demographic characteristics of mother–infant pairs. Mean 7S.D. Mothers Age at birth of infant (years) Race Turkish Other Education University High School Primary/Secondary School Occupation Working Not working Number of Delivery by Mothers 1 2 3 4 Smoking Habit Occupational exposure to lead Blood lead at delivery (mg/dL) Blood iron at delivery (mg/L)
Infants Gender Male Female Gestational age (weeks) Birth weights (kg) Birth length (cm) Head circumferences (cm) Placental weights (g) Umbilical cord blood lead (mg/dL) Placenta lead (mg/dL)
2. Materials and methods 2.1. Study subjects The study population was included 93 mother–newborn pairs. The women in this study consisted of consecutive cases coming to
457
n
S.D.: Standard deviation.
n (%)
29.5 7 4.8 93 (100) 0 (0) 48 (51.6) 32 (34.4) 13 (14.0) 55 (59.1) 38 (40.9) 48 (51.6) 33 (35.5) 10 (10.8) 2 (2.1) 0 (0) 0 (0) 3.20 7 1.26 346.18 7 91.37
48 (51.6) 45 (48.4) 39w1d 7 1w0.2d 3.337 0.46 49.747 1.98 35.297 1.15 629.89 7 149.80 2.577 0.97 8.80 7 3.75
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contamination. Each bag was marked with the subject's identification code and stored in a polyethylene bag at 20 °C in the Ankara University Analytical Toxicology Laboratory. 2.3. Determination of the HFE H63D SNP by the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method Genomic DNA was isolated from 100-μl whole blood samples using a Qiagen QIAamp DNA Mini Kit, according to the manufacturer's instructions. The H63D rs1799945 SNP in the HFE gene was genotyped using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. In order to screen for the HFE gene H63D polymorphism, a 294-bp fragment was amplified by PCR with the following primers: forward: 5′-ACATGGTTAAGGCCTGTTGC-3′ and reverse: 5′-CTTGCTGTGGTTGTGATTTTCC3′. Amplification was conducted on a Techne Tc 512 PCR System in a 50-μl reaction mixture containing 200 μM of dNTPs, 10 pmol each of the forward (F) and reverse (R) primers, 1 U of Hot Star Taq DNA polymerase (Qiagen), 10 PCR buffer (Qiagen) and 50 ng of genomic DNA. The PCR cycling conditions consisted of an initial denaturation step at 94 °C for 10 min; 35 cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min; and a final extension step at 72 °C for 5 min. The PCR product (294 bp) was then digested with MboI (New England Biolabs, Hertfordshire, UK) and incubated at 37 °C overnight. Digestion of the PCR product by MboI yields fragments that represent the presence of the H allele (138, 99 and 57 bp fragments) and the D allele (237 and 57 bp fragments). The undigested and digested polymerase chain reaction products were separated by gel electrophoresis on a 2.5% agarose gel, visualized by ethidium bromide staining under an ultraviolet illuminator, and then scanned and photographed using the Syngene Monitoring System. Results of RFLP for each variant in 30 randomly selected samples were confirmed by DNA sequencing method using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit on an ABI Prism 3100 Genetic Analyzer. The automated DNA sequencing was employed to confirm the authenticity of the amplified PCR products. 2.4. Determination of lead levels for maternal and cord blood samples and placenta tissues Prior to analysis, all placental samples were washed with 0.01% Triton X-100 solution and then three times with distilled water in order to prevent any contamination originating from maternal blood and mucus. Then, six representative samples were cut from each placenta using titanium tools, excluding the chorionic plate and decidua basalis. Two samples were taken from the center, avoiding the umbilical cord insertion, and four samples were taken from within 3 cm of the outer placental margin between the central region and the periphery. Each sample was dried for 24 h at 75 °C and weighed. Dried placenta samples and 1 ml of blood samples were dissolved in 10 ml of nitric acid in teflon microwave tubes and digested at 800 W and 220 °C for 20 min in a CEM Mars Xpress microwave oven. Afterwards, these digested solutions were diluted with 25 ml deionized water in 50-ml polypropylene tubes and Pb levels of placenta tissues, maternal and cord blood samples were quantified using Varian AA 240 Z Zeeman Graphite Atomic Absorption Spectrometry (GFAAS). 2.5. Statistical analyses The Statistical Package for Social Sciences (SPSS) version 16.0 software for Windows was used for the statistical analyses. The frequencies of H63D alleles and genotypes were obtained by direct
count, and the departure from the Hardy–Weinberg equilibrium was evaluated by the χ2 test. In the exploratory analysis, data showed a normal distribution (by Kolmogorow–Smirnow test); therefore, parametric Student's t-test was used in order to compare two independent groups in terms of metric variables. Categorical variables were compared by the χ2 test. Linear regression analysis was used to evaluate the association between variables. Since blood and placental lead levels found to be normally distributed, they were not transformed for regression analysis. Beta coefficients and 95% confidence intervals were calculated and given. Beta coefficients from regression analysis represent the change in placental and umbilical cord blood lead for 1% change in maternal blood lead. Maternal blood iron levels, gestational ages, birth weights, birth length, head circumferences, placental weights and socioeconomic status were assessed for potential confounding of the association between maternal blood lead and placental lead or umbilical cord blood lead. Pearson correlation was also used to determine whether there were relationships between gestational age and maternal blood lead and iron levels. Data were shown as mean 7standard deviation (S.D.). p o0.05 was considered as statistically significant.
3. Results Descriptive characteristics for mothers and infants are given in Table 1. The mean age of 93 healthy mothers was 29.57 4.8 years (ranging from 18 to 40 years). There was no history of occupational exposure to lead or smoking habit in any of the participants. Whole placentas were collected from the mothers who delivered at term neonates (mean gestational ages 39w1d 71w0.2d) with normal birth weights (mean 3.33 70.46 kg), birth length (mean 49.74 71.98 cm), head circumferences (mean 35.29 71.15 cm) and placental weights (mean 629.89 7149.80 g). The correlation between gestational age and maternal blood lead and iron levels was also examined with Pearson correlation. However, a statistically significant correlations were not found between gestational age and blood iron and lead level (r ¼0.069, p ¼0.512; r ¼0.121, p¼ 0.247, respectively). Also, there was not a significant correlation between maternal blood lead and iron levels (r ¼0.194, p ¼0.06). When maternal blood iron levels were examined according to HFE H63D genotypes, it was observed that blood iron levels were higher in mothers with HD þDD genotypes (348.95 7113.51 mg/L) than those with HH genotype (345.277 83.79 mg/L), but it was not statistically significant (p4 0.05). The HFE H63D genotype frequencies of mothers were found as 75.3% homozygote typical (HH), 23.6% heterozygote (HD) and 1.1% homozygote atypical (DD). Distribution of DD genotype in HFE H63D gene polymorphism was observed at very low frequency (only one mother). Therefore, HD and DD genotypes were gathered and all comparisons were made between HH (n ¼70) and HDþ DD (n ¼23) genotypes. The mean levels of lead in all maternal blood, placenta and umbilical cord blood samples and according to HFE H63D genotypes are given in Table 2. The placental tissue, maternal and umbilical cord blood lead levels of mothers with HD þDD genotypes were significantly higher than those with HH genotype (p o0.05). As a results of linear regression analysis (Table 3), a 1% increase in maternal blood lead is predicted to increase placental lead by 0.74% in infants whose mothers with HD þDD (β ¼0.74; CI 0.13, 0.31; p ¼0.001), whereas a 1% increase in maternal blood lead is expected to increase placental lead by 0.20% in infants whose mothers with HH (β ¼0.20; CI 0.01, 0.12; p ¼0.096). Regarding to umbilical cord blood lead, a 1% increase in maternal blood lead is predicted to increase umbilical cord blood lead by 0.49% in infants
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Table 2 HFE H63D polymorphism and lead concentrations of maternal bloods, umbilical cord bloods and dry placentas. Genotypes of H63D polymorphism
HH (n ¼70) HD þ DD (n ¼23) Total (n¼ 93) n p Value n
Maternal blood Pb levels (mg/dL)
Umbilical cord blood Pb levels (mg/dL)
Placental tissue Pb levels (mg/kg)
Mean 7S.D.
Minimum
Maximum
Mean 7 S.D.
Minimum
Maximum
Mean 7 S.D.
Minimum
Maximum
3.03 71.06 3.69 71.67 3.20 71.26 0.029
0.40 1.36 0.40
5.3 7.76 7.76
2.25 7 0.73 3.56 7 0.97 2.577 0.97 0.001
0.91 1.45 0.91
3.32 5.19 5.19
7.977 2.84 11.32 7 4.96 8.80 7 3.75 0.001
3.51 4.69 3.51
17.42 25.21 25.21
p Value in the table reflects the comparison of lead levels in HH vs. HDþ DD.
Table 3 Predicted influence of maternal blood lead on umbilical cord blood lead and placental lead. Maternal blood lead
β-Coefficients
95% CI
p-Value
HH
Umbilical cord blood lead Placental lead
0.04 0.20
0.14, 0.20 0.01, 0.12
0.736 0.096
HD þ DD
Umbilical cord blood lead Placental lead
0.49 0.74
0.06, 0.51 0.13, 0.31
0.018 0.001
Total
Umbilical cord blood lead Placental lead
0.31 0.50
0.08, 0.39 0.09, 0.20
0.003 0.001
whose mothers with HD þDD (β ¼0.49; CI 0.06, 0.51; p ¼0.018), whereas a 1% increase in maternal blood lead is expected to increase umbilical cord blood lead by 0.04% in infants whose mothers with HH (β ¼0.04; CI 0.14, 0.20; p ¼0.736). Our regression analysis predicts that for women with blood lead 5 mg/dL (CDC reference level), placental lead will be 1 mg/dL and umbilical cord blood lead will be 0.2 mg/dL in the infant born to the wild type mother and placental lead will be 3.7 mg/dL and umbilical cord blood lead will be 2.45 mg/dL in the infant born to the mother carrying the HFE H63D variant. The association between maternal blood lead and placental lead or umbilical cord blood lead was not modified by maternal blood iron levels, gestational ages, birth weights, birth length, head circumferences, placental weights or socioeconomic status.
4. Discussion Previously, the association between HFE gene variants and lead exposure was studied in children, elderly men or mixed aged groups (Hopkins et al., 2008; Barton et al., 1994; Wright et al., 2004). Hopkins et al. (2008) found 11% higher blood lead levels in Mexican children with HFE variant genotype compared with wildtype subjects. Barton et al. (1994) found higher blood lead levels in subjects with HH (from children to young/middle aged adults) compared with normal controls. In contrast, Wright et al. (2004) found lower bone/blood lead levels in elderly men with HFE variants. In Turkish population, the frequency of HFE H63D polymorphism was studied in blood donors (Simsek et al., 2004; Bozkaya et al., 2004) and in women with breast cancer (GunelOzcan et al., 2006). On the other hand, the association between HFE gene variants (C282Y and H63D) and lead exposure in mother–infant pairs was first studied by Karwowski et al. (2014) and maternal HFE C282Y gene variant status was found to be associated with placental lead transfer. Umbilical cord blood lead levels were detected to be lower in infants born to mothers with HFE C282Y variant than those with wild-type genotype. The association between umbilical cord blood lead and maternal blood lead was not modified by maternal HFE H63D genotypes. There are some differences between Karwowski et al.'s study and ours. In Karwowski et al.’s study conducted in northeastern Oklahoma,
placental lead levels were not measured and, thus, the effect of HFE H63D variant status on placental lead levels could not be examined. Therefore, the current study is the first to detect the effect of maternal HFE H63D gene polymorphism on lead levels in a triad of mother–placenta–fetus in a Turkish population. Higher blood lead levels in HFE H63D variant mothers in the current study indicated that the newborns of mothers with HFE H63D gene variants may be at risk of maternal exposure to lead. Furthermore, a significant association between maternal and umbilical cord blood lead level was detected in mothers with HD þDD, but not in mothers with HH. Thus, genetically susceptible people may not be fully protected in spite of the fact that the environmental and occupational lead exposures have been limited by health-based guidelines. Although it is suggested that HFE H63D gene carriers are susceptible to increased lead absorption, underlying mechanisms have not been exactly understood. In a study, in which knockout mice that lack the gene for HFE were used, an increase in transferrin saturation and iron storage in hepatocytes was observed (Waheed et al., 1999). In another study using HFE gene knockout mice, the expression of DMT-1 protein in the duodenum was found to be increased (Cantonwine et al., 2010; Onalaja and Claudio, 2000). The up-regulation of DMT-1 results in the increased absorption of iron and DMT-1 has affinity for several divalent cations such as lead and cadmium. Thus, there is a competition between iron and the toxic metals at the binding site of the transporter. Consistently, our findings indicated that blood lead levels of mothers with HDþ DD genotypes for H63D polymorphism were higher than those of HH genotype. Lead was also detected in blood samples of mothers with HH genotypes for HFE in spite of the fact that this study comprised healthy and occupationally unexposed women. This suggested that accumulated lead in pregnant women's bone can also be a source of internal lead exposure and can transfer from mother to fetus through placenta. Lead-free gasoline has been used in the Turkey markets since 1990s. When considering the mean ages of studied mothers (29.5 74.8 years), they were children in 1990s and may be exposed to lead especially by air. After ingesting, lead is first taken up by red blood cells and, then, it is deposited in the bones with time. Lead can be mobilized from bone along with calcium during pregnancy and lactation due to increased demands for calcium. According to the isotopic studies, during pregnancy, the skeletal contribution to blood lead levels increases from 9% to 65% (Gomaa et al., 2002). In the current study, maternal blood lead was linearly associated with both placental lead (β ¼0.50; CI 0.09, 0.20; p ¼0.001) and umbilical cord blood lead (β ¼0.31; CI 0.08, 0.39; p ¼0.003) in all 93 triad of mother–placenta–fetus (Table 3), which confirmed lead's trans-placental transfer. A 1% increase in maternal blood lead is predicted to increase placental lead by 0.50% and umbilical cord blood lead by 0.31%, indicating that placenta reduces passage of a number of lead to the fetus. When linear regression analysis were repeated according to HFE H63D genotypes, maternal blood lead was linearly associated with placental lead (β ¼0.74; CI 0.13,
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0.31; p ¼0.001) and umbilical cord blood lead (β ¼ 0.49; CI 0.06, 0.51; p ¼0.018) among infants born mothers with HD þDD genotypes, whereas this association was not significant among infants born mothers with HH genotype. Taken together, it may be suggested that maternal HFE H63D polymorphism seems to have an effect not only on lead level in maternal blood but also on transfer of lead from placenta to fetus. In parallel line with our suggestion, Karwowski et al. (2014) reported that maternal genotype primarily affects the lead’s trans-placental transfer, whereas fetal HFE C282Y and H63D gene variants do not alter the lead transfer across the placenta (Karwowski et al., 2014). However, in contrast to Karwowski et al. (2014), we found that maternal blood lead was linearly associated with umbilical cord blood in HFE H63D variant mother–infant pairs. Unlike HFE C282Y polymorphism, how H63D polymorphism in the HFE gene modifies iron absorption has not been exactly understood. The effects of C282Y and H63D mutations on HFE protein may be different, and, thus, their effects on placental lead transfer in wild-type and variants was found to be different in Karwowski et al.'s study and ours. In a previous study by Balesaria et al. (2012), it was found that maternal HFE status plays a role in the transfer of iron across the placenta by modulating of gene expressions in the placenta. According to this study, when the mother was a HFE-knockout mouse, the expression of TfR, DMT-1 and ferroportin elevated in the placenta (Balesaria et al., 2012). We hypothesized that lead found in maternal circulation might transfer to fetal circulation by competing with iron due to the increased DMT-1 in the placenta. So far, there has been no evidence that ferroportin is involved in lead transport. However, it was reported that lead, like iron, can bind to human transferrin that can transport lead into the cell by interacting with TfR (Luo et al., 2011). Based on this knowledge, the significant placental transfer in mothers with HD þDD genotype is probably due to increased expression of DMT1 and TfR in the placenta. However, further investigations are necessary with different techniques to improve this hypothesis. Although maternal blood lead was not linearly associated with neither placental lead nor umbilical cord blood lead in mothers with HFE H63D wild-type genotype (p ¼0.096 and p ¼0.736, respectively), it was obvious that a number of lead can cross the placenta in these mothers since lead was also detected in placentas and cord bloods of infants born to these mothers. The United States Center for Disease Control (CDC) accepted the 10 mg/dL blood lead level as allowable threshold limit (CDC, 1991). In the present study, none of the biological samples exceeded this limit. On the other hand, mean maternal blood lead level (3.20 mg/ dL) in the current study seems higher than some other countries including Sweden (1.139 mg/dL), Taiwan (1.58 mg/dL), Brazil (1.736 mg/dL), France (0.018 mg/dL) and South Africa (2.3 mg/dL). This might be related to ancient lead mines located in Turkey (Fujihara et al., 2009). Similar to maternal blood, the mean cord blood lead in the current study (2.57 mg/dL) was higher than those reported in Montreal (1.7 mg/dL), New York (1.6 mg/dL), South Africa (1.54 mg/dL), Belgium (1.47 mg/dL) and Taiwan (1.29 mg/dL). In conclusion, the present study indicated for the first time that Turkish mothers with H63D gene variants had higher lead levels of their newborn's umbilical cord blood and their unborn child may be at increased risk of internal exposure to lead as compared to wild-type counterparts. Thus, we suggested that maternal HFE H63D status may have an effect not only on increased maternal blood lead levels but also on lead transfer from maternal circulation to fetal circulation across the placenta. However, to test this hypothesis, further investigations are necessary with different study design and techniques.
Disclosure summary The author has nothing to declare.
Conflict of interest The author has nothing to declare.
Acknowledgements This study was funded by the Ankara University Scientific Research Projects Coordination Unit (BAP; Project Number: 12H5150001).
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Maternal hemochromatosis gene H63D single-nucleotide polymorphism and lead levels of placental tissue, maternal and umbilical cord blood Zeliha Kayaalti a,n, Dilek Kaya-Akyüzlü a, Esma Söylemez a,b, Tülin Söylemezoğlu a a b
Ankara University, Institute of Forensic Sciences, Ankara, Turkey Middle Black Sea Passage Generation of Agricultural Research Station Director, Tokat, Turkey
art ic l e i nf o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 30 April 2015 Accepted 5 May 2015
Human hemochromatosis protein (HFE), a major histocompatibility complex class I-like integral membrane protein, participates in the down regulation of intestinal iron absorption by binding to transferrin receptor (TR). HFE competes with transferrin-bound iron for the TR and thus reduces uptake of iron into cells. On the other hand, a lack of HFE increases the intestinal absorption of iron similarly to iron deficiency associated with increasing in absorption and deposition of lead. During pregnancy, placenta cannot prevent transfer lead to the fetus; even low-level lead poisoning causes neurodevelopmental toxicity in children. The aim of this study was to determine the association between the maternal HFE H63D single-nucleotide polymorphism and lead levels in placental tissue, maternal blood and umbilical cord bloods. The study population comprised 93 mother–placenta pairs. Venous blood from mother was collected to investigate lead levels and HFE polymorphism that was detected by standard PCR–RFLP technique. Cord bloods and placentas were collected for lead levels which were analyzed by dual atomic absorption spectrometer system. The HFE H63D genotype frequencies of mothers were found as 75.3% homozygote typical (HH), 23.6% heterozygote (HD) and 1.1% homozygote atypical (DD). Our study results showed that the placental tissue, umbilical cord and maternal blood lead levels of mothers with HDþ DD genotypes were significantly higher than those with HH genotype (po 0.05). The present study indicated for the first time that mothers with H63D gene variants have higher lead levels of their newborn's placentas and umbilical cord bloods. & 2015 Elsevier Inc. All rights reserved.
Keywords: Human hemochromatosis protein Single nucleotide polymorphism Maternal blood Umbilical cord blood Placental tissue Lead
1. Introduction Iron is an essential and also vital trace element that is a component of many hemoproteins and non-heme iron proteins. It plays a critical role in oxygen sensing and transport, electron transfer, and catalysis. However, when present in excess and not tightly bound, it can be toxic by catalyzing the production of oxygen radicals. Thus, the levels of iron in the body are tightly controlled to prevent either iron depletion or iron overload by several proteins such as transferrin, divalent metal transporter-1 (DMT-1), ferroportin, hemochromatosis (HFE) and hepcidin (Horvathova et al., 2010; Cantonwine et al., 2010; Rolfs et al., 2002). The human HFE protein participating in the down regulation of iron absorption is a major histocompatibility complex class I-like integral membrane protein which is expressed in crypt cells of duodenum and syncytiotrophoblast cells of placenta (Rolfs et al., n
2002; Waheed et al., 1999; Akesson et al., 2000; Hanson et al., 2001). HFE does not bind or transport iron, but rather competes with iron-loaded transferrin for binding to the transferrin receptor (TfR) (Rolfs et al., 2002; Townsend and Drakesmith, 2002). HFE gene first identified as HLAH by Feder et al. in 1996 is located at 6p21.3 and covers approximately 10 kilobases and almost 4.6 megabases telomeric from HLA-A (Hanson et al., 2001). Among 37 allelic variants of this gene, the commonly found two variants C282Y and H63D (Hanson et al., 2001; Wang et al., 2007) are associated with hereditary hemochromatosis (HH) that is an autosomal recessive blood disorder characterized by an increase in iron absorption by comparison to iron excretion (Rolfs et al., 2002; Wang et al., 2007; Elmrghni et al., 2011). Elevated iron absorption is supposed to go along with increased absorption of chemically related divalent metals such as lead (Elsenhans et al., 2011). Although lead is a naturally occurring heavy metal, a local and
Correspondence to: Institute of Forensic Sciences, Ankara University, Dikimevi, 06590 Ankara, Turkey. Fax: þ90 312 3192077. E-mail address: [email protected] (Z. Kayaalti).
http://dx.doi.org/10.1016/j.envres.2015.05.004 0013-9351/& 2015 Elsevier Inc. All rights reserved.
Z. Kayaalti et al. / Environmental Research 140 (2015) 456–461
more diffuse lead has been enriched in the indoor and outdoor environments due to industrial development, urbanization and lifestyle factors and, thus, lead exposure becomes one of the most important health hazards (Bierkens et al., 2011). The main sources of lead contamination in soil and lead pollution in air are leaded gasoline and dust from lead-based house paints. Other sources are cigarette smoke, lead smelting industries, fuels used for heating, battery recycling plants and occupational exposure in automotive and aircraft paint manufacturing (Dorea, 2004). Lead, having no beneficial role to humans, can be received through food, contaminated air and dust, and accumulates particularly in the bones with a half-life of around 30 years (D’Souza et al., 2003; Tekin et al., 2012). Lead has adverse effects to mainly renal, hematopoietic and neurologic systems. The most susceptible populations to the adverse neurodevelopmental effects of lead are children, particularly infants in the neonatal period and fetus since neurodevelopment begins in this period and the blood–brain barrier is still immature (Goyer, 1990; Goyer, 1997; Molina et al., 2011). Moreover, exposure levels that do not harm the mother can be fetotoxic because of the differences between the many biochemical pathways of the adult and the fetus (Baghurst et al., 1991; Al-Saleh et al., 2011). Fetal exposure to lead begins at the 21st week of pregnancy and continues throughout the life (Goyer, 1990). Although the placenta, an interface between the developing fetus and the mother, has mechanisms that restrict the entry of toxicants (Al-Saleh et al., 2011), it cannot protect the fetus from exposure to lead from the mother's blood. In experimental models, it was demonstrated that umbilical cord blood flow rate affected the transfer of lead linearly (Goyer, 1990). Furthermore, iron deficiency or dietary iron consumption of the mother can also alter lead uptake (Molina et al., 2011). Hence, genes related to iron metabolism could modify lead absorption and pregnant women with genetic variants affecting iron metabolism could have children at increased risk of exposure to lead. To test this hypothesis, this study aimed to examine the effect of maternal HFE H63D single-nucleotide polymorphism (SNP) on mother–placenta–fetus lead levels. HFE gene was selected since (a) HFE influences the expression of other metal transporters such as DMT1 and ferroportin; (b) HFE is one of the genes that are recognized to influence lead toxicokinetics and toxicodynamics; and, (c) HFE protein is expressed at the apical membrane of syncytiotrophoblasts and placental iron transfer occurs at this site. Previously, Karwowski et al. (2014) studied the effect of maternal and/or infant hemochromatosis (both HFE C282Y and H63D) gene variants on maternal blood lead (MBL) and umbilical cord blood lead (UCBL). In this study, no statistically significant differences between geometric mean blood lead value and variant status was detected. Placental transfer of lead was found to be associated with only maternal HFE C282Y gene variant status, but not with maternal HFE H63D variants. Compared to Karwowski's study, we examined the effect of maternal HFE H63D (rs1799945; Gene ID:3077, Accession number: NP_001287678) gene variant status on lead levels of maternal and cord blood samples as well as placental tissues in Turkish population. Thus, to the best of our knowledge, this is the first study indicating that maternal HFE H63D gene variant status was significantly associated with lead levels of placental tissue and maternal and cord blood samples and may modify the placental transfer of lead in a triad of mother–placenta–fetus.
the Gynecology Department of Ankara University's Faculty of Medicine, from February to October in 2011. Mothers with gestational ages Z36 weeks (n ¼127) were screened in the hospital and asked to fill out a questionnaire, which included medical and dietary history, as well as data on occupational and potential environmental sources of metal exposure, and socioeconomic status. A small questionnaire used to gather demographic information was also given to individuals. Only Turkish subjects were included in the study. Healthy, non-smoking, non-anemic mothers (n ¼93) living in Ankara for more than 3 years without a history of alcohol, drug use or chronic disease were included in the study. Ineligibility criteria was a medical history of renal failure, diabetes, carcinoma, diagnosed hepatic or cardiovascular diseases that may be related to possible heavy metal accumulation from environmental or occupational exposures. Thirty-four women were excluded from the study due to occupational exposure (n ¼2, printing workers), smoking habit (n ¼25) and a medical history of chronic diseases (n ¼7). Written informed consent was obtained from those eligible for the study. Infant characteristics such as gestational age, birth weight, birth length and head circumference were also recorded (Table 1). Placenta, maternal and cord blood samples were collected at delivery by cesarean section or spontaneous labor. This study was approved by the institutional ethics committee (approval no: 33-730 in 2011). 2.2. Sample collection Two ml of venous blood was taken from each woman before delivery and from the umbilical cord during delivery into tubes with EDTA and stored at 4 °C. Placentas were collected and placed in a plastic bag immediately after delivery to avoid external metal Table 1 Demographic characteristics of mother–infant pairs. Mean 7S.D. Mothers Age at birth of infant (years) Race Turkish Other Education University High School Primary/Secondary School Occupation Working Not working Number of Delivery by Mothers 1 2 3 4 Smoking Habit Occupational exposure to lead Blood lead at delivery (mg/dL) Blood iron at delivery (mg/L)
Infants Gender Male Female Gestational age (weeks) Birth weights (kg) Birth length (cm) Head circumferences (cm) Placental weights (g) Umbilical cord blood lead (mg/dL) Placenta lead (mg/dL)
2. Materials and methods 2.1. Study subjects The study population was included 93 mother–newborn pairs. The women in this study consisted of consecutive cases coming to
457
n
S.D.: Standard deviation.
n (%)
29.5 7 4.8 93 (100) 0 (0) 48 (51.6) 32 (34.4) 13 (14.0) 55 (59.1) 38 (40.9) 48 (51.6) 33 (35.5) 10 (10.8) 2 (2.1) 0 (0) 0 (0) 3.20 7 1.26 346.18 7 91.37
48 (51.6) 45 (48.4) 39w1d 7 1w0.2d 3.337 0.46 49.747 1.98 35.297 1.15 629.89 7 149.80 2.577 0.97 8.80 7 3.75
458
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contamination. Each bag was marked with the subject's identification code and stored in a polyethylene bag at 20 °C in the Ankara University Analytical Toxicology Laboratory. 2.3. Determination of the HFE H63D SNP by the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method Genomic DNA was isolated from 100-μl whole blood samples using a Qiagen QIAamp DNA Mini Kit, according to the manufacturer's instructions. The H63D rs1799945 SNP in the HFE gene was genotyped using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. In order to screen for the HFE gene H63D polymorphism, a 294-bp fragment was amplified by PCR with the following primers: forward: 5′-ACATGGTTAAGGCCTGTTGC-3′ and reverse: 5′-CTTGCTGTGGTTGTGATTTTCC3′. Amplification was conducted on a Techne Tc 512 PCR System in a 50-μl reaction mixture containing 200 μM of dNTPs, 10 pmol each of the forward (F) and reverse (R) primers, 1 U of Hot Star Taq DNA polymerase (Qiagen), 10 PCR buffer (Qiagen) and 50 ng of genomic DNA. The PCR cycling conditions consisted of an initial denaturation step at 94 °C for 10 min; 35 cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min; and a final extension step at 72 °C for 5 min. The PCR product (294 bp) was then digested with MboI (New England Biolabs, Hertfordshire, UK) and incubated at 37 °C overnight. Digestion of the PCR product by MboI yields fragments that represent the presence of the H allele (138, 99 and 57 bp fragments) and the D allele (237 and 57 bp fragments). The undigested and digested polymerase chain reaction products were separated by gel electrophoresis on a 2.5% agarose gel, visualized by ethidium bromide staining under an ultraviolet illuminator, and then scanned and photographed using the Syngene Monitoring System. Results of RFLP for each variant in 30 randomly selected samples were confirmed by DNA sequencing method using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit on an ABI Prism 3100 Genetic Analyzer. The automated DNA sequencing was employed to confirm the authenticity of the amplified PCR products. 2.4. Determination of lead levels for maternal and cord blood samples and placenta tissues Prior to analysis, all placental samples were washed with 0.01% Triton X-100 solution and then three times with distilled water in order to prevent any contamination originating from maternal blood and mucus. Then, six representative samples were cut from each placenta using titanium tools, excluding the chorionic plate and decidua basalis. Two samples were taken from the center, avoiding the umbilical cord insertion, and four samples were taken from within 3 cm of the outer placental margin between the central region and the periphery. Each sample was dried for 24 h at 75 °C and weighed. Dried placenta samples and 1 ml of blood samples were dissolved in 10 ml of nitric acid in teflon microwave tubes and digested at 800 W and 220 °C for 20 min in a CEM Mars Xpress microwave oven. Afterwards, these digested solutions were diluted with 25 ml deionized water in 50-ml polypropylene tubes and Pb levels of placenta tissues, maternal and cord blood samples were quantified using Varian AA 240 Z Zeeman Graphite Atomic Absorption Spectrometry (GFAAS). 2.5. Statistical analyses The Statistical Package for Social Sciences (SPSS) version 16.0 software for Windows was used for the statistical analyses. The frequencies of H63D alleles and genotypes were obtained by direct
count, and the departure from the Hardy–Weinberg equilibrium was evaluated by the χ2 test. In the exploratory analysis, data showed a normal distribution (by Kolmogorow–Smirnow test); therefore, parametric Student's t-test was used in order to compare two independent groups in terms of metric variables. Categorical variables were compared by the χ2 test. Linear regression analysis was used to evaluate the association between variables. Since blood and placental lead levels found to be normally distributed, they were not transformed for regression analysis. Beta coefficients and 95% confidence intervals were calculated and given. Beta coefficients from regression analysis represent the change in placental and umbilical cord blood lead for 1% change in maternal blood lead. Maternal blood iron levels, gestational ages, birth weights, birth length, head circumferences, placental weights and socioeconomic status were assessed for potential confounding of the association between maternal blood lead and placental lead or umbilical cord blood lead. Pearson correlation was also used to determine whether there were relationships between gestational age and maternal blood lead and iron levels. Data were shown as mean 7standard deviation (S.D.). p o0.05 was considered as statistically significant.
3. Results Descriptive characteristics for mothers and infants are given in Table 1. The mean age of 93 healthy mothers was 29.57 4.8 years (ranging from 18 to 40 years). There was no history of occupational exposure to lead or smoking habit in any of the participants. Whole placentas were collected from the mothers who delivered at term neonates (mean gestational ages 39w1d 71w0.2d) with normal birth weights (mean 3.33 70.46 kg), birth length (mean 49.74 71.98 cm), head circumferences (mean 35.29 71.15 cm) and placental weights (mean 629.89 7149.80 g). The correlation between gestational age and maternal blood lead and iron levels was also examined with Pearson correlation. However, a statistically significant correlations were not found between gestational age and blood iron and lead level (r ¼0.069, p ¼0.512; r ¼0.121, p¼ 0.247, respectively). Also, there was not a significant correlation between maternal blood lead and iron levels (r ¼0.194, p ¼0.06). When maternal blood iron levels were examined according to HFE H63D genotypes, it was observed that blood iron levels were higher in mothers with HD þDD genotypes (348.95 7113.51 mg/L) than those with HH genotype (345.277 83.79 mg/L), but it was not statistically significant (p4 0.05). The HFE H63D genotype frequencies of mothers were found as 75.3% homozygote typical (HH), 23.6% heterozygote (HD) and 1.1% homozygote atypical (DD). Distribution of DD genotype in HFE H63D gene polymorphism was observed at very low frequency (only one mother). Therefore, HD and DD genotypes were gathered and all comparisons were made between HH (n ¼70) and HDþ DD (n ¼23) genotypes. The mean levels of lead in all maternal blood, placenta and umbilical cord blood samples and according to HFE H63D genotypes are given in Table 2. The placental tissue, maternal and umbilical cord blood lead levels of mothers with HD þDD genotypes were significantly higher than those with HH genotype (p o0.05). As a results of linear regression analysis (Table 3), a 1% increase in maternal blood lead is predicted to increase placental lead by 0.74% in infants whose mothers with HD þDD (β ¼0.74; CI 0.13, 0.31; p ¼0.001), whereas a 1% increase in maternal blood lead is expected to increase placental lead by 0.20% in infants whose mothers with HH (β ¼0.20; CI 0.01, 0.12; p ¼0.096). Regarding to umbilical cord blood lead, a 1% increase in maternal blood lead is predicted to increase umbilical cord blood lead by 0.49% in infants
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459
Table 2 HFE H63D polymorphism and lead concentrations of maternal bloods, umbilical cord bloods and dry placentas. Genotypes of H63D polymorphism
HH (n ¼70) HD þ DD (n ¼23) Total (n¼ 93) n p Value n
Maternal blood Pb levels (mg/dL)
Umbilical cord blood Pb levels (mg/dL)
Placental tissue Pb levels (mg/kg)
Mean 7S.D.
Minimum
Maximum
Mean 7 S.D.
Minimum
Maximum
Mean 7 S.D.
Minimum
Maximum
3.03 71.06 3.69 71.67 3.20 71.26 0.029
0.40 1.36 0.40
5.3 7.76 7.76
2.25 7 0.73 3.56 7 0.97 2.577 0.97 0.001
0.91 1.45 0.91
3.32 5.19 5.19
7.977 2.84 11.32 7 4.96 8.80 7 3.75 0.001
3.51 4.69 3.51
17.42 25.21 25.21
p Value in the table reflects the comparison of lead levels in HH vs. HDþ DD.
Table 3 Predicted influence of maternal blood lead on umbilical cord blood lead and placental lead. Maternal blood lead
β-Coefficients
95% CI
p-Value
HH
Umbilical cord blood lead Placental lead
0.04 0.20
0.14, 0.20 0.01, 0.12
0.736 0.096
HD þ DD
Umbilical cord blood lead Placental lead
0.49 0.74
0.06, 0.51 0.13, 0.31
0.018 0.001
Total
Umbilical cord blood lead Placental lead
0.31 0.50
0.08, 0.39 0.09, 0.20
0.003 0.001
whose mothers with HD þDD (β ¼0.49; CI 0.06, 0.51; p ¼0.018), whereas a 1% increase in maternal blood lead is expected to increase umbilical cord blood lead by 0.04% in infants whose mothers with HH (β ¼0.04; CI 0.14, 0.20; p ¼0.736). Our regression analysis predicts that for women with blood lead 5 mg/dL (CDC reference level), placental lead will be 1 mg/dL and umbilical cord blood lead will be 0.2 mg/dL in the infant born to the wild type mother and placental lead will be 3.7 mg/dL and umbilical cord blood lead will be 2.45 mg/dL in the infant born to the mother carrying the HFE H63D variant. The association between maternal blood lead and placental lead or umbilical cord blood lead was not modified by maternal blood iron levels, gestational ages, birth weights, birth length, head circumferences, placental weights or socioeconomic status.
4. Discussion Previously, the association between HFE gene variants and lead exposure was studied in children, elderly men or mixed aged groups (Hopkins et al., 2008; Barton et al., 1994; Wright et al., 2004). Hopkins et al. (2008) found 11% higher blood lead levels in Mexican children with HFE variant genotype compared with wildtype subjects. Barton et al. (1994) found higher blood lead levels in subjects with HH (from children to young/middle aged adults) compared with normal controls. In contrast, Wright et al. (2004) found lower bone/blood lead levels in elderly men with HFE variants. In Turkish population, the frequency of HFE H63D polymorphism was studied in blood donors (Simsek et al., 2004; Bozkaya et al., 2004) and in women with breast cancer (GunelOzcan et al., 2006). On the other hand, the association between HFE gene variants (C282Y and H63D) and lead exposure in mother–infant pairs was first studied by Karwowski et al. (2014) and maternal HFE C282Y gene variant status was found to be associated with placental lead transfer. Umbilical cord blood lead levels were detected to be lower in infants born to mothers with HFE C282Y variant than those with wild-type genotype. The association between umbilical cord blood lead and maternal blood lead was not modified by maternal HFE H63D genotypes. There are some differences between Karwowski et al.'s study and ours. In Karwowski et al.’s study conducted in northeastern Oklahoma,
placental lead levels were not measured and, thus, the effect of HFE H63D variant status on placental lead levels could not be examined. Therefore, the current study is the first to detect the effect of maternal HFE H63D gene polymorphism on lead levels in a triad of mother–placenta–fetus in a Turkish population. Higher blood lead levels in HFE H63D variant mothers in the current study indicated that the newborns of mothers with HFE H63D gene variants may be at risk of maternal exposure to lead. Furthermore, a significant association between maternal and umbilical cord blood lead level was detected in mothers with HD þDD, but not in mothers with HH. Thus, genetically susceptible people may not be fully protected in spite of the fact that the environmental and occupational lead exposures have been limited by health-based guidelines. Although it is suggested that HFE H63D gene carriers are susceptible to increased lead absorption, underlying mechanisms have not been exactly understood. In a study, in which knockout mice that lack the gene for HFE were used, an increase in transferrin saturation and iron storage in hepatocytes was observed (Waheed et al., 1999). In another study using HFE gene knockout mice, the expression of DMT-1 protein in the duodenum was found to be increased (Cantonwine et al., 2010; Onalaja and Claudio, 2000). The up-regulation of DMT-1 results in the increased absorption of iron and DMT-1 has affinity for several divalent cations such as lead and cadmium. Thus, there is a competition between iron and the toxic metals at the binding site of the transporter. Consistently, our findings indicated that blood lead levels of mothers with HDþ DD genotypes for H63D polymorphism were higher than those of HH genotype. Lead was also detected in blood samples of mothers with HH genotypes for HFE in spite of the fact that this study comprised healthy and occupationally unexposed women. This suggested that accumulated lead in pregnant women's bone can also be a source of internal lead exposure and can transfer from mother to fetus through placenta. Lead-free gasoline has been used in the Turkey markets since 1990s. When considering the mean ages of studied mothers (29.5 74.8 years), they were children in 1990s and may be exposed to lead especially by air. After ingesting, lead is first taken up by red blood cells and, then, it is deposited in the bones with time. Lead can be mobilized from bone along with calcium during pregnancy and lactation due to increased demands for calcium. According to the isotopic studies, during pregnancy, the skeletal contribution to blood lead levels increases from 9% to 65% (Gomaa et al., 2002). In the current study, maternal blood lead was linearly associated with both placental lead (β ¼0.50; CI 0.09, 0.20; p ¼0.001) and umbilical cord blood lead (β ¼0.31; CI 0.08, 0.39; p ¼0.003) in all 93 triad of mother–placenta–fetus (Table 3), which confirmed lead's trans-placental transfer. A 1% increase in maternal blood lead is predicted to increase placental lead by 0.50% and umbilical cord blood lead by 0.31%, indicating that placenta reduces passage of a number of lead to the fetus. When linear regression analysis were repeated according to HFE H63D genotypes, maternal blood lead was linearly associated with placental lead (β ¼0.74; CI 0.13,
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0.31; p ¼0.001) and umbilical cord blood lead (β ¼ 0.49; CI 0.06, 0.51; p ¼0.018) among infants born mothers with HD þDD genotypes, whereas this association was not significant among infants born mothers with HH genotype. Taken together, it may be suggested that maternal HFE H63D polymorphism seems to have an effect not only on lead level in maternal blood but also on transfer of lead from placenta to fetus. In parallel line with our suggestion, Karwowski et al. (2014) reported that maternal genotype primarily affects the lead’s trans-placental transfer, whereas fetal HFE C282Y and H63D gene variants do not alter the lead transfer across the placenta (Karwowski et al., 2014). However, in contrast to Karwowski et al. (2014), we found that maternal blood lead was linearly associated with umbilical cord blood in HFE H63D variant mother–infant pairs. Unlike HFE C282Y polymorphism, how H63D polymorphism in the HFE gene modifies iron absorption has not been exactly understood. The effects of C282Y and H63D mutations on HFE protein may be different, and, thus, their effects on placental lead transfer in wild-type and variants was found to be different in Karwowski et al.'s study and ours. In a previous study by Balesaria et al. (2012), it was found that maternal HFE status plays a role in the transfer of iron across the placenta by modulating of gene expressions in the placenta. According to this study, when the mother was a HFE-knockout mouse, the expression of TfR, DMT-1 and ferroportin elevated in the placenta (Balesaria et al., 2012). We hypothesized that lead found in maternal circulation might transfer to fetal circulation by competing with iron due to the increased DMT-1 in the placenta. So far, there has been no evidence that ferroportin is involved in lead transport. However, it was reported that lead, like iron, can bind to human transferrin that can transport lead into the cell by interacting with TfR (Luo et al., 2011). Based on this knowledge, the significant placental transfer in mothers with HD þDD genotype is probably due to increased expression of DMT1 and TfR in the placenta. However, further investigations are necessary with different techniques to improve this hypothesis. Although maternal blood lead was not linearly associated with neither placental lead nor umbilical cord blood lead in mothers with HFE H63D wild-type genotype (p ¼0.096 and p ¼0.736, respectively), it was obvious that a number of lead can cross the placenta in these mothers since lead was also detected in placentas and cord bloods of infants born to these mothers. The United States Center for Disease Control (CDC) accepted the 10 mg/dL blood lead level as allowable threshold limit (CDC, 1991). In the present study, none of the biological samples exceeded this limit. On the other hand, mean maternal blood lead level (3.20 mg/ dL) in the current study seems higher than some other countries including Sweden (1.139 mg/dL), Taiwan (1.58 mg/dL), Brazil (1.736 mg/dL), France (0.018 mg/dL) and South Africa (2.3 mg/dL). This might be related to ancient lead mines located in Turkey (Fujihara et al., 2009). Similar to maternal blood, the mean cord blood lead in the current study (2.57 mg/dL) was higher than those reported in Montreal (1.7 mg/dL), New York (1.6 mg/dL), South Africa (1.54 mg/dL), Belgium (1.47 mg/dL) and Taiwan (1.29 mg/dL). In conclusion, the present study indicated for the first time that Turkish mothers with H63D gene variants had higher lead levels of their newborn's umbilical cord blood and their unborn child may be at increased risk of internal exposure to lead as compared to wild-type counterparts. Thus, we suggested that maternal HFE H63D status may have an effect not only on increased maternal blood lead levels but also on lead transfer from maternal circulation to fetal circulation across the placenta. However, to test this hypothesis, further investigations are necessary with different study design and techniques.
Disclosure summary The author has nothing to declare.
Conflict of interest The author has nothing to declare.
Acknowledgements This study was funded by the Ankara University Scientific Research Projects Coordination Unit (BAP; Project Number: 12H5150001).
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