RESEARCH ARTICLE
Placental Cadmium Levels Are Associated with Increased Preeclampsia Risk Jessica E. Laine1, Paul Ray2, Wanda Bodnar2, Peter H. Cable2, Kim Boggess3, Steven Offenbacher4, Rebecca C. Fry2* 1 Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, United States of America, 2 Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, United States of America, 3 Department of Obstetrics and Gynecology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America, 4 Department of Periodontology, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, United States of America * [email protected]
Abstract OPEN ACCESS Citation: Laine JE, Ray P, Bodnar W, Cable PH, Boggess K, Offenbacher S, et al. (2015) Placental Cadmium Levels Are Associated with Increased Preeclampsia Risk. PLoS ONE 10(9): e0139341. doi:10.1371/journal.pone.0139341 Editor: Jaymie Meliker, Stony Brook University, Graduate Program in Public Health, UNITED STATES Received: May 29, 2015 Accepted: September 11, 2015 Published: September 30, 2015 Copyright: © 2015 Laine et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Environmental exposure to heavy metals is a potentially modifiable risk factor for preeclampsia (PE). Toxicologically, there are known interactions between the toxic metal cadmium (Cd) and essential metals such as selenium (Se) and zinc (Zn), as these metals can protect against the toxicity of Cd. As they relate to preeclampsia, the interaction between Cd and these essential metals is unknown. The aims of the present study were to measure placental levels of Cd, Se, and Zn in a cohort of 172 pregnant women from across the southeast US and to examine associations of metals levels with the odds of PE in a nested casecontrol design. Logistic regressions were performed to assess odds ratios (OR) for PE with exposure to Cd controlling for confounders, as well as interactive models with Se or Zn. The mean placental Cd level was 3.6 ng/g, ranging from 0.52 to 14.5 ng/g. There was an increased odds ratio for PE in relationship to placental levels of Cd (OR = 1.5; 95% CI: 1.1– 2.2). The Cd-associated OR for PE increased when analyzed in relationship to lower placental Se levels (OR = 2.0; 95% CI: 1.1–3.5) and decreased with higher placental Se levels (OR = 0.98; 95% CI: 0.5–1.9). Similarly, under conditions of lower placental Zn, the Cdassociated OR for PE was elevated (OR = 1.8; 95% CI: 0.8–3.9), whereas with higher placental Zn it was reduced (OR = 1.3; 95% CI: 0.8–2.0). Data from this pilot study suggest that essential metals may play an important role in reducing the odds of Cd-associated preeclampsia and that replication in a larger cohort is warranted.
Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by grants from the National Institutes of Health (http://www.nih. gov): R01 ES019315, P42 ES005948, and 5U01DE014577. The funders of this study had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction Cadmium (Cd) is a ubiquitous toxic metal currently ranked 7th on the priority List of Hazardous Substances by the Agency for Toxic Substances and Disease Registry [1]. Industrial activity such as non-ferrous metal mining and refining, manufacturing and application of phosphate fertilizers, fossil fuel combustion, and waste increases Cd leaching into the soil/water/air/food
PLOS ONE | DOI:10.1371/journal.pone.0139341 September 30, 2015
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Placental Cadmium and Preeclampsia
[2]. Cd can contaminate the soil/water/air/food by disposal of consumer products such as batteries, pigments, coatings, and plastics. Another source of Cd exposure is via cigarette smoke [2]. Chronic Cd exposure is associated with renal disease, hypertension, and cancer [3]. Cd exposure during pregnancy has the potential to harm both the mother and fetus, as the placenta does not serve as a complete barrier against Cd [4]. In support of this, maternal serum Cd levels are significantly associated with umbilical and neonatal Cd levels highlighting the transfer of this toxic metal to the fetus [5–7]. In utero exposure to Cd is associated with low birth weight, decreased neonatal length and head circumference, and impaired childhood neurobehavioral and physiological development [8]. In addition, mean placental levels of Cd have been shown to be higher in preeclamptics versus normotensive women [9]. However, the relationship between placental Cd levels and risk for preeclampsia is currently unknown. There are known interactions between Cd and essential metals such as zinc (Zn) and selenium (Se). Specifically, Zn and Se are both anti-oxidants that protect against Cd-induced toxicity [10]. Also, increased placental Cd concentrations may impair essential elements such as Se and Zn transfer to the fetus [4]. This is of concern as Zn deficiency is associated with fetal growth restriction and neurodevelopment abnormalities [11, 12]. Furthermore, maternal Zn deficiency has been associated with preeclampsia [13]. Women with preeclampsia have been shown to have lower levels of Se compared to normotensive women [14–16]. The relationship between Cd, Se, and Zn levels in the placenta as they relate to of risk of preeclampsia is not known. The aims of the present study were to measure placental Cd, Se, and Zn levels using samples from a pregnancy cohort representing women across the southeastern US and to examine associations of placental metal levels with the risk of preeclampsia. Biomonitoring of maternal exposure to metals will potentially help to reduce prenatal exposures to toxic metals and inform nutritional supplementation of essential metals, ultimately reducing future adverse pregnancy complications and birth outcomes.
Materials and Methods Study Population This study is a nested case-control study within the Maternal Oral Therapy to Reduce Obstetric Risk (MOTOR) study. This study was approved by the University of North Carolina, Chapel Hill Institutional Review Board. The MOTOR cohort was a randomized, treatment-masked, controlled clinical trial of 1,806 pregnant women with periodontal disease who were receiving standard obstetric care (ClinicalTrials.gov Identifier: NCT00097656) [17]. Written consent was obtained from all participants. The cohort has been fully described previously [17]. Briefly, from December 2003 through October 2007 subjects were enrolled at Duke University Medical Center (and the affiliated clinic at Lincoln Health Center), the University of Alabama at Birmingham Medical Center, and two obstetric sites of the University of Texas Health Science Center at San Antonio (University Health Center-Downtown of University Health Systems and Salinas Clinic of the San Antonio Metropolitan Health District). Randomization occurred between February 2004 and September 2007. For this nested case-control study, 172 subjects were selected; 86 with preeclampsia and 86 healthy, normotensive women. Preeclampsia was defined using criteria current at the time of the original study [18] as newly diagnosed hypertension (blood pressure >140/90 mmHg on two occasions 6 hours apart) and proteinuria (>300 mg of protein in a 24 h urine collection or urine protein/creatinine ratio of 0.3 mg/dL) occurring at > 20 weeks’ gestation. Controls were randomly chosen among those without preeclampsia. Baseline maternal characteristics such as
PLOS ONE | DOI:10.1371/journal.pone.0139341 September 30, 2015
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Placental Cadmium and Preeclampsia
education, socioeconomic status (SES), and age, amongst others did not differ between the nested cohort and the parent cohort (data not shown).
Placental Collection and Metals Measurements Immediately after placental delivery, a single, full-thickness biopsy of the placenta was obtained and flash-frozen. Biopsies were taken from the central zone of the placenta and standardized across all samples as previously described [17]. Abnormal gross placental pathology (i.e. infarcted areas) was avoided. Harvested placental tissues were stored at -70°C. Placenta samples (169.7 ± 33.1 mg) were digested with 70% nitric acid at room temperature for five hours before being placed in an 85 degree heating block overnight. After cooling to room temperature, 30% hydrogen peroxide was added and the samples were returned to the heating block. The vials were removed, cooled, and vented every twenty minutes for the first hour. Digestion continued for an additional four hours and was complete after 24 hours. The samples were then were diluted to 4 mL with deionized water. The determination of total Cd, Se, and Zn was performed using the Agilent Technologies 7500cx inductively coupled plasma mass spectrometer (ICP-MS), (Santa Clara, CA. USA). Analytes were quantified using published protocols [19]. Cd and Zn isotopes measured were 111 m/z and 66 m/z respectively. External calibration and quality control standards were prepared from National Institute Standards Technology (NIST) traceable solutions (High Purity Standards, Charleston, SC. USA). The detection limit reported was corrected for dilution and normalized to a typical sample mass of 0.150g. The limit of detection for the metals was: 1.5 ng/g (Cd), 1.5 ng/g (Se), and 300 ng/g (Zn).
Statistical Analyses The statistical package SAS 9.4 (SAS Institute Inc., Cary, North Carolina) was used to analyze the data. Spearman rank correlations were calculated to measure the inter-relationship among placental metals levels and were considered to be significantly correlated if p
Placental Cadmium Levels Are Associated with Increased Preeclampsia Risk Jessica E. Laine1, Paul Ray2, Wanda Bodnar2, Peter H. Cable2, Kim Boggess3, Steven Offenbacher4, Rebecca C. Fry2* 1 Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, United States of America, 2 Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, United States of America, 3 Department of Obstetrics and Gynecology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America, 4 Department of Periodontology, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, United States of America * [email protected]
Abstract OPEN ACCESS Citation: Laine JE, Ray P, Bodnar W, Cable PH, Boggess K, Offenbacher S, et al. (2015) Placental Cadmium Levels Are Associated with Increased Preeclampsia Risk. PLoS ONE 10(9): e0139341. doi:10.1371/journal.pone.0139341 Editor: Jaymie Meliker, Stony Brook University, Graduate Program in Public Health, UNITED STATES Received: May 29, 2015 Accepted: September 11, 2015 Published: September 30, 2015 Copyright: © 2015 Laine et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Environmental exposure to heavy metals is a potentially modifiable risk factor for preeclampsia (PE). Toxicologically, there are known interactions between the toxic metal cadmium (Cd) and essential metals such as selenium (Se) and zinc (Zn), as these metals can protect against the toxicity of Cd. As they relate to preeclampsia, the interaction between Cd and these essential metals is unknown. The aims of the present study were to measure placental levels of Cd, Se, and Zn in a cohort of 172 pregnant women from across the southeast US and to examine associations of metals levels with the odds of PE in a nested casecontrol design. Logistic regressions were performed to assess odds ratios (OR) for PE with exposure to Cd controlling for confounders, as well as interactive models with Se or Zn. The mean placental Cd level was 3.6 ng/g, ranging from 0.52 to 14.5 ng/g. There was an increased odds ratio for PE in relationship to placental levels of Cd (OR = 1.5; 95% CI: 1.1– 2.2). The Cd-associated OR for PE increased when analyzed in relationship to lower placental Se levels (OR = 2.0; 95% CI: 1.1–3.5) and decreased with higher placental Se levels (OR = 0.98; 95% CI: 0.5–1.9). Similarly, under conditions of lower placental Zn, the Cdassociated OR for PE was elevated (OR = 1.8; 95% CI: 0.8–3.9), whereas with higher placental Zn it was reduced (OR = 1.3; 95% CI: 0.8–2.0). Data from this pilot study suggest that essential metals may play an important role in reducing the odds of Cd-associated preeclampsia and that replication in a larger cohort is warranted.
Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by grants from the National Institutes of Health (http://www.nih. gov): R01 ES019315, P42 ES005948, and 5U01DE014577. The funders of this study had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction Cadmium (Cd) is a ubiquitous toxic metal currently ranked 7th on the priority List of Hazardous Substances by the Agency for Toxic Substances and Disease Registry [1]. Industrial activity such as non-ferrous metal mining and refining, manufacturing and application of phosphate fertilizers, fossil fuel combustion, and waste increases Cd leaching into the soil/water/air/food
PLOS ONE | DOI:10.1371/journal.pone.0139341 September 30, 2015
1/9
Placental Cadmium and Preeclampsia
[2]. Cd can contaminate the soil/water/air/food by disposal of consumer products such as batteries, pigments, coatings, and plastics. Another source of Cd exposure is via cigarette smoke [2]. Chronic Cd exposure is associated with renal disease, hypertension, and cancer [3]. Cd exposure during pregnancy has the potential to harm both the mother and fetus, as the placenta does not serve as a complete barrier against Cd [4]. In support of this, maternal serum Cd levels are significantly associated with umbilical and neonatal Cd levels highlighting the transfer of this toxic metal to the fetus [5–7]. In utero exposure to Cd is associated with low birth weight, decreased neonatal length and head circumference, and impaired childhood neurobehavioral and physiological development [8]. In addition, mean placental levels of Cd have been shown to be higher in preeclamptics versus normotensive women [9]. However, the relationship between placental Cd levels and risk for preeclampsia is currently unknown. There are known interactions between Cd and essential metals such as zinc (Zn) and selenium (Se). Specifically, Zn and Se are both anti-oxidants that protect against Cd-induced toxicity [10]. Also, increased placental Cd concentrations may impair essential elements such as Se and Zn transfer to the fetus [4]. This is of concern as Zn deficiency is associated with fetal growth restriction and neurodevelopment abnormalities [11, 12]. Furthermore, maternal Zn deficiency has been associated with preeclampsia [13]. Women with preeclampsia have been shown to have lower levels of Se compared to normotensive women [14–16]. The relationship between Cd, Se, and Zn levels in the placenta as they relate to of risk of preeclampsia is not known. The aims of the present study were to measure placental Cd, Se, and Zn levels using samples from a pregnancy cohort representing women across the southeastern US and to examine associations of placental metal levels with the risk of preeclampsia. Biomonitoring of maternal exposure to metals will potentially help to reduce prenatal exposures to toxic metals and inform nutritional supplementation of essential metals, ultimately reducing future adverse pregnancy complications and birth outcomes.
Materials and Methods Study Population This study is a nested case-control study within the Maternal Oral Therapy to Reduce Obstetric Risk (MOTOR) study. This study was approved by the University of North Carolina, Chapel Hill Institutional Review Board. The MOTOR cohort was a randomized, treatment-masked, controlled clinical trial of 1,806 pregnant women with periodontal disease who were receiving standard obstetric care (ClinicalTrials.gov Identifier: NCT00097656) [17]. Written consent was obtained from all participants. The cohort has been fully described previously [17]. Briefly, from December 2003 through October 2007 subjects were enrolled at Duke University Medical Center (and the affiliated clinic at Lincoln Health Center), the University of Alabama at Birmingham Medical Center, and two obstetric sites of the University of Texas Health Science Center at San Antonio (University Health Center-Downtown of University Health Systems and Salinas Clinic of the San Antonio Metropolitan Health District). Randomization occurred between February 2004 and September 2007. For this nested case-control study, 172 subjects were selected; 86 with preeclampsia and 86 healthy, normotensive women. Preeclampsia was defined using criteria current at the time of the original study [18] as newly diagnosed hypertension (blood pressure >140/90 mmHg on two occasions 6 hours apart) and proteinuria (>300 mg of protein in a 24 h urine collection or urine protein/creatinine ratio of 0.3 mg/dL) occurring at > 20 weeks’ gestation. Controls were randomly chosen among those without preeclampsia. Baseline maternal characteristics such as
PLOS ONE | DOI:10.1371/journal.pone.0139341 September 30, 2015
2/9
Placental Cadmium and Preeclampsia
education, socioeconomic status (SES), and age, amongst others did not differ between the nested cohort and the parent cohort (data not shown).
Placental Collection and Metals Measurements Immediately after placental delivery, a single, full-thickness biopsy of the placenta was obtained and flash-frozen. Biopsies were taken from the central zone of the placenta and standardized across all samples as previously described [17]. Abnormal gross placental pathology (i.e. infarcted areas) was avoided. Harvested placental tissues were stored at -70°C. Placenta samples (169.7 ± 33.1 mg) were digested with 70% nitric acid at room temperature for five hours before being placed in an 85 degree heating block overnight. After cooling to room temperature, 30% hydrogen peroxide was added and the samples were returned to the heating block. The vials were removed, cooled, and vented every twenty minutes for the first hour. Digestion continued for an additional four hours and was complete after 24 hours. The samples were then were diluted to 4 mL with deionized water. The determination of total Cd, Se, and Zn was performed using the Agilent Technologies 7500cx inductively coupled plasma mass spectrometer (ICP-MS), (Santa Clara, CA. USA). Analytes were quantified using published protocols [19]. Cd and Zn isotopes measured were 111 m/z and 66 m/z respectively. External calibration and quality control standards were prepared from National Institute Standards Technology (NIST) traceable solutions (High Purity Standards, Charleston, SC. USA). The detection limit reported was corrected for dilution and normalized to a typical sample mass of 0.150g. The limit of detection for the metals was: 1.5 ng/g (Cd), 1.5 ng/g (Se), and 300 ng/g (Zn).
Statistical Analyses The statistical package SAS 9.4 (SAS Institute Inc., Cary, North Carolina) was used to analyze the data. Spearman rank correlations were calculated to measure the inter-relationship among placental metals levels and were considered to be significantly correlated if p
