Article pubs.acs.org/est
Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Umbilical Cord Concentrations of Selected Heavy Metals and Risk for Orofacial Clefts Wenli Ni,† Wenlei Yang,† Jinhui Yu,† Zhiwen Li,† Lei Jin,† Jufen Liu,† Yali Zhang,† Linlin Wang,*,† and Aiguo Ren† †
Institute of Reproductive and Child Health, NHC Key Laboratory of Reproductive Health, Department of Epidemiology and Biostatistics, School of Public Health, Peking University Health Science Center, Peking University, Beijing 100191, China
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ABSTRACT: Although arsenic (As), cadmium (Cd), lead (Pb), and nickel (Ni) have the ability to induce orofacial clefts (OFCs) in rodents, evidence is absent from human epidemiological investigations with markers of in utero exposure. We investigated the associations between concentrations of As, Cd, Pb, and Ni in umbilical cord tissues and risk of OFCs, and the interactions between each pair of metals on OFC risk in a case-control study. Umbilical cord concentrations of metals were determined by inductively coupled plasma mass spectrometry in 92 OFC cases and 200 nonmalformed controls. Concentrations above the median of all subjects was associated with an elevated OFC risk of 8.36-fold for As, 7.22-fold for Cd, 15.32-fold for Pb, and 6.79-fold for Ni. Concentrations of As, Cd, Pb, and Ni were also associated with risks for OFC subtypes. When metal concentrations were divided into tertiles by levels of metal concentrations of all subjects, dose−response relationships of risks for total OFCs and subtypes with As, Cd, Pb, and Ni concentrations were demonstrated. Significant synergistic interaction between As and Ni on the risk of OFCs was also observed. Therefore, elevated in utero exposure to As, Cd, Pb, and Ni may increase the risks of OFCs in newborns.
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INTRODUCTION Orofacial clefts (OFCs) are one of some common congenital anomalies that have a significant impact on health and quality of life in humans.1−3 OFCs derive from an embryopathy with the tissues of the lip or palate not joining properly.2 The etiologies of OFCs are generally thought to be multifactorial, involving both genetic and environmental factors.4 Some environmental factors, including maternal exposure to PM2.5, agricultural pesticide, dioxins, and tobacco smoking, may increase the risk of OFCs.5−8 Some heavy metals with adverse effects on human health, especially on human reproduction, are a concern; however, the role of heavy metals in the occurrence of OFCs remains largely unclear. Heavy metals are omnipresent in the environment, occurring in varying concentrations in air, bedrock, soil, water, and all biological matter.9 Arsenic (As), cadmium (Cd), lead (Pb), and nickel (Ni) are well-known heavy metals that have been an important public health concern because the environmental levels have increased due to continued worldwide anthropogenic activities.10,11 It has been established that As, Cd, Pb, and Ni can cross the placental barrier resulting in in utero exposures to the developing fetus.12−15 Experimental studies have shown that As, Cd, Pb, and Ni exposures during pregnancy affect embryogenesis and offspring development in mice or rat.12,16−18 Additionally, As, Cd, Pb, and Ni were found to have teratogenicity such as OFCs, exencephaly, eye defects, skeletal deformities, or heart defects in mice, rat, fish, or hamster.19−23 However, epidemiological evidence for the association of OFC risks is absent with in utero exposure to heavy metals, and limited maternal exposure to heavy metals. © XXXX American Chemical Society
One epidemiological study with a small sample size (20 controls and 40 cases) reported higher hair As levels compared to hair Pb and Cd among mothers of infants with OFCs, relative to control mothers.24 Studies based on occupational exposure assessments reported that maternal or parental exposure to Pb at work is a risk factor for OFCs.25,26 No study investigated the effect of maternal or in utero exposure to Ni on the development of OFCs in humans. The umbilical cord is derived from the yolk sac and the urine sac, which is part of the fetus. Compared with external exposure and maternal internal exposure such as pollutant concentrations in maternal hair, the internal in utero exposure, such as pollutant concentrations in umbilical cord is more likely to assess the real levels of pollutant exposure in fetus.27 Previous study has used umbilical cord concentrations of heavy metals as biomarkers of fetal exposure to investigate the exposure to trace elements and minerals and risk of early childhood wheezing and eczema.28 However, up to now no studies have investigated the association between umbilical cord concentrations of heavy metals, as a measure of internal exposure and risks of OFCs in humans. Therefore, in the present study, we aimed to examine the associations between umbilical cord concentrations of As, Cd, Pb, and Ni and the risks of OFCs in a case-control study in northern China.29 Previous studies reported significant Received: Revised: Accepted: Published: A
May 5, 2018 August 8, 2018 August 22, 2018 August 22, 2018 DOI: 10.1021/acs.est.8b02404 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Umbilical Cord Tissue Collection and Laboratory Assessment. Samples of umbilical cord were collected immediately after delivery, placed in polyethylene bags, kept at −20 °C, and transferred on dry ice to our laboratory for processing and analyses. Prior to analysis, approximately 2.0 g of umbilical cord samples were separated from the total umbilical cord tissues using scissors. The separated umbilical cord samples were rinsed three times with deionized water to remove cord blood and dried with clean tissue paper to remove excess water, and then freeze-dried. The scissors used to prepare umbilical cord tissue was repeatedly sterilized with an alcohol solution between every umbilical cord sample in order to avoid contamination of the samples. Subsequently, approximately 0.200 g of dry umbilical cord tissues were weighed (accurate to 0.001 g), and then digested in a solution of 3 mL of nitric acid (UP-grade) and 0.5 mL of H2O2 in a high-pressure microwave digestion system from room temperature to 160 °C for 5 min, 160−200 °C for 5 min, and 200 °C for 20 min, 1300 W, 40 bar (Ultra WAVE, Milestone, Italy) with quartz vessels. After digestion, the samples were brought to a final volume of 15 mL with high-purity deionized water. Finally, the samples were diluted with ultrapure water at a ratio of 1:4. A blank solution was prepared and carried through the preparation and digestion procedure alongside each of 14 samples, in order to check the possible contamination during the digestion procedure and sample manipulation. Total As, Cd, Pb, and Ni concentrations were determined by an ICP-MS (7700x, Agilent, USA). Certified standards of Chinese national reference materials (GSB 04-1714-2004 for As, GSB 041721-2004 for Cd, GSB 04-1742-2004 for Pb, and GSB 041740-2004 for Ni) were used in the calibration and validation of the standard curves. All calibration straight lines had correlation coefficients >0.999. This system was calibrated using rhodium (GSB 04-1745-2004) as the internal reference. To check the stability of the process, a sample of standard materials made from pig liver (GBW10051), which had known reference concentrations of As, Cd, Pb, and Ni was prepared and analyzed in parallel for each twentieth umbilical cord samples. ICP-MS measurements were finished for cases and controls in random sequence at the meantime for each of the four metals, and operators were masked to the group of the sample. The limits of detection (LOD) for As, Cd, Pb, and Ni were 0.010 ng/mL, 0.001 ng/mL, 0.002 ng/mL, and 0.016 ng/ mL, respectively. The concentrations of As, Cd, Pb, and Ni were all above the LOD for the samples in our analyses. Statistical Methods. The differences in demographic and lifestyle characteristics between the case and control group were evaluated by Chi-square test or Fisher’s exact test. The median with interquartile range was used to describe the skewed distributions. Differences of metal contents between cases and controls were evaluated using the Mann−Whitney U test, and the metal concentrations of different gestational weeks in the cases were evaluated by the Kruskal−Wallis test. Because no clinical dose threshold was available a priori as the reference of cutoff value for transformation of categorical variable, metal concentrations were dichotomized using the median of all subjects as the cutoff value in association analysis, or classified into tertiles by levels of metal concentrations of all subjects in dose−response analysis. Risk was estimated by the use of odds ratio (OR), and the precision of each OR was assessed by its 95% confidence internal (CI). The odds of an event is a ratio of the frequency (or likelihood) of its
interactions between different metal exposures on health outcomes,30 so we further assessed the possible interaction effects among umbilical cord As, Cd, Pb, and Ni on risks for OFCs.
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MATERIALS AND METHODS Study Design and Subjects. Study subjects were recruited based on a case−control study of birth defects in five counties (Pingding, Xiyang, Shouyang, Taigu, and Zezhou) of Shanxi province in northern China, which has been described elsewhere.29 Concisely, a surveillance system of birth defects was established in the study area in 2002. The surveillance system monitored major external structural birth defects through active case ascertainment. County or township health workers verified the diagnoses by physical examination of the fetal body for all pregnancy outcomes and filled out a reporting form for each case. When a case infant was identified, a healthy newborn without congenital malformations was selected as a control, matched to the case by newborn sex, mother’s county of residence, and the date of mother’s last menstrual period (±4 months). Although the study was originally designed as a matched case−control study, some umbilical cord samples were not available because consent could not be obtained from some subjects; therefore, the matched case-control pairs were broken and the unmatched case-control design was adopted in the present study. To maximize the sample size and the statistical power, we selected all OFC cases with available umbilical cord samples and all controls with available umbilical cord samples, collected between 2003 and 2016 in this study. A total of 92 OFC cases [51 with cleft lip with cleft palate (CLP), 37 with cleft lip only (CLO), and 4 with cleft palate only (CPO)] and 200 controls were included. Previous studies suggested that CLP and CLO have distinct etiologies,31,32 so we analyzed them separately. The study was approved by the Institutional Review Board of Peking University. All participants provided written informed consent. Previous human (maternal exposure, external exposure) and animal studies have found that As, Cd, Pb, Ni, and methylmercury exposure may increase the risk of OFCs.19,21,23−25,33,34 However, up to now no studies have investigated the association between umbilical cord concentrations of heavy metals, as a measure of internal exposure and risks of OFCs in humans. We detected As, Cd, Pb, and Ni, which can be simultaneously detected by inductively coupled plasma mass spectrometry (ICP-MS), to analyze the association between As, Cd, Pb, and Ni and risks of OFCs in the present study. We gave up the assay of total mercury by ICP-MS because of a “memory effect” in the detecting process, which often generates inaccurate results. Additionally, methylmercury, which is one of the most toxic forms of mercury, cannot be detected by the ICP-MS method. Further, when enough umbilical cord samples are collected, we will use the cold atomic fluorescence spectrometry method to detect levels of methylmercury. Data Collection. Trained local health care workers conducted face-to-face interviews with mothers of cases and controls before the mothers were discharged from hospitals, mostly within 1 week of delivery or termination of pregnancies, to collect the exposure information using a structured questionnaire, including demographic characteristics, reproductive history, lifestyle behaviors, folic acid supplementation, from 1 month before through 2 months after conception. B
DOI: 10.1021/acs.est.8b02404 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology occurrence to the frequency (or likelihood) of its nonoccurrence. The OR is a comparison of the odds of an event after exposure to a risk factor with the odds of that event in a control situation, used to indicate the intensity of the association between exposure and disease in a case-control study.35 We used the unconditional logistic regression and Chisquare trend test to evaluate the relationships between As, Cd, Pb, and Ni concentrations and risks for OFCs and OFC subtypes, respectively. Unconditional logistic regression analysis was used to estimate the adjusted OR (aOR), adjusting for maternal age, body mass index (BMI), education, occupation, parity, history of pregnancy affected by birth defects, folic acid supplementation, maternal smoking exposure (active or passive smoking), alcohol drinking, and gestational weeks. Correlations between umbilical cord metals were evaluated using spearman correlation. We also examined possible interaction between each pair of metals on risks for OFCs by producing a product between the two dichotomous variables in the logistic model, respectively. Three sensitivity analyses were conducted to assess robustness of our results. First, we conducted a sensitivity analysis using metals as continuous variables and compared the results with categorical variables. Second, we conducted sensitivity analysis limited to nonsyndromic orofacial clefts without other malformations. Third, to examine the impact of different sample sizes, we randomly selected 92 controls from all controls (200) to analyze the association between As, Cd, Pb, and Ni, and risk of OFCs. We considered P < 0.05 statistically significant. All data were analyzed using SPSS 23.0 software (SPSS, Chicago, IL, USA).
Table 1. Characteristics of Women Who Had Pregnancies Affected by OFCs (Cases) and Women Who Delivered Healthy Infants (Controls) n(%)a characteristics
controls (n = 200)
maternal age (years)
