Metals and hypertensive disorders of pregnancy: MIREC Study (Article 1)

pregnancy: MIREC Study (Article 1)

Short title: metals and hypertensive disorders in pregnancy

Louopou R. Camara, Helen Trottier, William D. Fraser

Statut de l’article : Le manuscrit est en correction à Santé Canada. Il doit être soumis au

journal Environmental Health Perspectives.

Contribution des auteurs :

- Louopou Rosalie Camara a constitué la base de données, elle a fait toutes les analyses et la rédaction du manuscrit sous la supervision de Dre Helen Trottier et de Dr William D. Fraser.

- Les coauteurs : Dre Helen Trottier a participé aux analyses et à la rédaction du manuscrit. Dr William D. Fraser un l’un des chercheurs principaux de l’étude MIREC. Ils ont participé au design de l'étude, à sa conduite et aux analyses ainsi qu'à la rédaction du manuscrit.

Abstract

Background: Metals have been associated with hypertensive disorders of pregnancy (HDP) but

data are scarce.

Objective: To assess the association between metals (arsenic, lead, cadmium, mercury, or

manganese) and HDP.

Methods: Urinary arsenic was measured in the first trimester and blood metals in the first and

third trimesters. Blood pressure was assessed by trimester in 2001 pregnant women recruited in 10 Canadian cities. Associations (adjusted odd ratios (aOR) and 95% Confidence intervals (CI)) between metals and HDP (gestational hypertension (GH) overall, GH and preeclampsia (PE)) were analyzed using logistic and multinomial regression models. Adjusted beta (aß) were also estimated using concurrent measures of metals and blood pressure to analyze the associationsusing linear generalized estimating equations

Results: First trimester blood manganese (> 9.89 versus <7.69 µg/l) was associated with lower

risks of GH overall (aOR = 0.68; 95% CI: 0.46, 0.99) and PE (aOR=0.48; 95% CI: 0.23, 0.98), especially in women delivering males babies. When measured concurrently, increasing blood manganese was associated with higher blood pressure (aß=1.52; 95% CI: 0.80-2.24). Third trimester blood mercury (> 0.82 versus <0.36 µg/l) was also associated with a lower risk of GH overall (aOR = 0.62; 95% CI: 0.39, 0.98). First trimester blood arsenic (> 0.97 versus 0.60 µg/l) was associated with an elevated risk of PE, adjusted for the other metals (aOR = 2.75; 95% CI 1.13, 6.73). No significant associations were observed for blood lead, cadmium or urinary arsenic.

Conclusion: Manganese and mercury were associated with lower odds of GH overall and/or PE

whereas blood arsenic was associated with an increased risk of PE. Further studies are required to replicate these results in similar populations.

Introduction

Manganese (Mn) is an essential element required for growth, development, and maintenance of health (Avila et al. 2013). It plays a leading role in the formation of enzymes, in particular pyruvate carboxylase, glutamine synthetase and superoxide dismutase (Avila et al. 2013; Wood 2009) which are respectively important in glucose metabolism, expressed in astrocytes (Avila et al. 2013) and antioxidant enzymes that protect cells from damage due to free radicals (Candas and Li 2014). Although an essential element, Mn toxicity can also result from high (Avila et al. 2013; Vigeh et al. 2013) as well as low levels of exposure (Avila et al. 2013; Sarwar et al. 2013). Arsenic (As), lead (Pb), cadmium (Cd) and mercury (Hg) have no known physiological function (Jaishankar et al. 2014). Their toxicity depends on the dose, route of exposure, chemical species, age, gender, genetics, and nutritional status of exposed individuals (Tchounwou et al. 2012).

Human exposure to these metals has increased due to the expansion of their use in several applications (Tchounwou et al. 2012). Although, food is the primary source of exposure to Mn, it can also be found in drinking water, air (especially occupational exposures), and soil (Health Canada 2016). Food is a major source of cadmium (Cd), arsenic (As) and mercury (Hg) followed by cigarette smoke for Cd, drinking water for As (Järup 2003) and dental amalgam for Hg (Health Canada 2004; Järup 2003). Lead (Pb) can be present in jewellery, toys, cosmetics, contaminated food, dust, water (WHO 2016) and air (Järup 2003).

Exposure to these metals may induce low nitric oxide levels, endothelial dysfunction and increased oxidative stress (Jomova et al. 2011; Lemos et al. 2012; Cordova et al. 2013; Vaziri 2008) or may impact on the functioning of matrix metalloproteinases (MMPs) (Au et al. 2016; Lacorte et al. 2015). These mechanisms have been associated with gestational hypertension

without preeclampsia (GH) (Tayebjee et al. 2005; Kennedy et al. 2012) or preeclampsia (PE) (Chen and Khalil 2017; Powe et al. 2011). Hypertension in pregnancy, which includes GH and PE (Magee et al. 2014), contributes to maternal morbidity (Nakimuli et al. 2016), mortality (Lo et al. 2013) and represents 3.6% to 9.1% of pregnancies in developed countries (Roberts et al. 2011). Approximately 50% of women with GH go on to develop PE (Leeman et al. 2016) which affects 2-8% of pregnancies (Duley 2009) and which is a major source of perinatal morbidity and mortality (Leeman et al. 2016).

Epidemiological studies have observed an association between these metals and hypertensive disorders of pregnancy (HDP). Depending on the form in which it is measured, an association has been reported between As and blood pressure among pregnant women (Farzan et al. 2015). Methylated forms in urine (monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) is generally excreted) (Drobna et al. 2009) and are thought to be less toxic (Jomova et al. 2011). The natural organic form, arsenobetaine, may have minimal toxic effects (Jomova et al. 2011; Suzuki et al. 2002). Studies have reported a relationship between Pb and both PE (Ikechukwu et al. 2012; Motawei et al. 2013) and GH (Yazbeck et al. 2009). Cd exposure has been linked to GH, especially among smokers (Kosanovic and Jokanovic 2007), although this finding is not consistent, as smoking (which is a source of metals (OMS 2012)), has also been associated with a reduced risk of PE (Wikström and Stephansson 2010).

Associations between Hg and GH have been reported in both occupational (Pan et al. 2007) and non-occupational settings (Wells et al. 2017). But the adverse effect of Hg may be antagonized by fish containing omega-3 fatty (Houston 2011) due to their antioxidative properties (Dasilva et al. 2017). Some studies have shown a relationship between Mn and GH

(Vigeh et al. 2013) or PE (Sarwar et al. 2013). Finally, male fetus has been associated with PE (Myers et al. 2015) and it is possible that the relationship between metals exposure (Mn, Pb, Cd, Hg) and hypertension may be modified by the fetal sex (Bushnik et al. 2014; Lee et al. 2015; Nielsen et al. 2012) and maternal smoking status (Kosanovic and Jokanovic 2007).

Few cohort studies have analyzed the association between metal exposures and HDP and there is inconsistency in the literature (Maduray et al. 2017; Mordukhovich et al. 2012; Mozaffarian et al. 2012; Yazbeck et al. 2009). Studies of Hg, As and Cd are especially scarce. One of the main objectives of the MIREC Study (Maternal-Infant Research on Environmental Chemicals) was to investigate whether exposure to metals during pregnancy was associated with higher risks of HDP.

Methods

Study design and population

The MIREC Study is a prospective cohort study of 2001 pregnant women recruited from 10 Canadian cities (Vancouver, Edmonton, Winnipeg, Sudbury, Ottawa, Kingston, Hamilton, Toronto, Montreal, and Halifax). This study took place over a 4-year recruitment period (2008- 2011). Women were eligible if they were in the first trimester of pregnancy without a history of significant medical complications, aged ≥18 years, and able to communicate in French or in English. Further details on the MIREC study population have been published previously (Arbuckle et al. 2013). After beginning the study, 18 women subsequently withdrew and asked that their data be destroyed, and 74 women were excluded because of miscarriage or stillbirth (including 3 cases of chronic hypertension and 1 case of preeclampsia), leaving a final sample size for this analysis of 1909 participants. The study was approved by Health Canada’s Research

Ethics Board and the Research Ethics Committee of Sainte-Justine University Hospital in Montreal, Quebec (Canada) as well as in all MIREC affiliated recruitment centers. All participants signed consent forms.

During each trimester, women participated in a clinic visit where they provided biospecimens, had physical measurements taken and completed questionnaires on sociodemographic and exposure characteristics. Clinical data were also abstracted from the medical charts at each visit.

Blood and urine metals

Maternal whole blood from the first and third trimesters of pregnancy was analyzed for As, Pb, Cd, Hg, and Mn and maternal urines from the first trimester were analyzed for arsenite, arsenate, arsenobetaine, MMA, DMA and specific gravity (Arbuckle et al. 2016; Ettinger et al. 2017). The blood measurements were performed by a single-quadrupole inductively coupled plasma mass spectrometry (ICP-MS) Elan DRC-II system (Perkin Elmer, Norwalk CT, USA) method and high performance liquid chromatography coupled with ICP-MS (Varian 820-MS, Varian Inc., Palo Alto CA, USA) was used for urinary speciated As measurements. The specific gravity was measured by refractometry (UG-1, Atago # 3461, Atago U.S.A. Inc., Bellevue WA, USA). All laboratory analyses were performed by the Centre de Toxicologie du Québec, Institut National de Santé Publique du Québec (INSPQ), Quebec, Canada.

Limits of detection (LOD) were: 0.2247 µg/l (3 nmol/l) for blood As, 0.1036 µg/dl (0.001 μmol/l) for Pb, 0.0454 µg/l (0.4 nmol/l) for Cd, 0.1204 µg/l (0.6 nmol/l) for Hg and 0.54945 µg/l (10 nmol/l) for Mn. For urinary speciated As, the limit of detection was 0.75 µg As/l (0.01 µmol As/l). In this study, a value equivalent to the half of LOD was attributed to blood and urine

concentrations below the LOD. In terms of blood concentrations, no metal had more than 12% below the LOD. For urinary measurements, values below LOD were 13.8% for DMA, 49.9% for arsenobetaine and over 80% for arsenite, arsenate and MMA. Arsenite, arsenate, MMA and DMA were summed under inorganic As. Total urinary speciated As was calculated by summing arsenite, arsenate, MMA, DMA and arsenobetaine.

Blood pressure and diagnosis of HDP

The maternal systolic (SBP) and diastolic (DBP) blood pressures were measured during each clinic visit by the staff using a sphygmomanometer. Two measures of blood pressure were taken about one minute apart and averaged for each visit. Blood pressure was assessed in a sitting position, with the cuffed arm resting on a desk at the level of the heart. The Korotkoff phase V (disappearance) was used for DBP measurement. The Korotkoff phase IV (muffling) was used only when a phase V was absent.

The mean arterial pressure of the two measures taken at each visit was determined for four time periods: visit 1 (6 to 13 weeks), visit 2 (16 to 21 weeks), visit 3 (32 to 34 weeks) and visit 4 (after admission for delivery). The results were used according to this formula: (SBP + 2 DBP)/3, obtaining one value for mean arterial pressure. Blood pressure data following admission for delivery were abstracted from the hospital chart. The diagnostic criteria for HDP were based on the Society of Obstetricians and Gynaecologists of Canada Guidelines (Magee et al. 2014). According to this guideline, a diagnosis of hypertension is based on the average of two measurements of SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg, taken at least 1 minute apart in MIREC study and using the same arm. De novo appearance of hypertension at ≥ 20 weeks of gestation with or without PE defined GH overall. The presence of proteinuria (proteinuria 24h ≥

300 mg/24h) or maternal complications defines PE whereas their absence defines GH without preeclampsia (GH in this study) (Magee et al. 2014). In this study, four categories were established for HDP status: Normotensive, GH overall and its subcategories GH without PE and PE. Gestational age (GA) in weeks was based on last menstrual period and/or early ultrasound result. An algorithm summarizing the steps for the diagnosis of HDP is available in supplemental material (see Fig. S1).

Covariates

Data on potential covariates were derived from questionnaires administered at the first, second and third visits as well as from medical chart reviews. The following variables were analyzed as covariates: maternal age at delivery in years (continuous form), parity (multiparous, nulliparous), ethnicity (caucasian, non-caucasian), body mass index before pregnancy (BMI) (weight in kg divided by height squared in meters and categorized as underweight, normal weight, overweight, obese), weight gain during pregnancy (kg) (difference between last weight measured prior to delivery and weight measured at first trimester visit), education (university, college, less than college), household income ($<15,000, 16,000-35,000, 36,000-45,000, 46,000-55,000, 56,000- 65,000, 66,000-75,000, 76,000-90,000, > 90,000), maternal smoking (never, quit before pregnancy, quit during pregnancy, current smoker), any alcohol consumption during the 3 months before visit 1 (no, yes), and fish (excluding shellfish) consumption (relative total quantity serving size per day calculated from the food frequency questionnaire by Morisset et al. (2016)). To account for the effect of urinary dilution, specific gravity was also considered as a covariate in models of urinary speciated As.

Descriptive statistics were performed to analyze participant characteristics according to HDP status using Student’s t-test, the Mann-Whitney, Kruskal-Wallis or chi-square tests. Geometric means (GM) (with 95% confidence intervals (CI)) and categorical distributions of metals were determined for each outcome category (normotensive, GH overall, GH and PE). Levels of metals were compared using Mann-Whitney and Kruskal-Wallis and tertiles were compared using the chi-square test. Spearman correlations were performed to test the correlations between first and third trimester blood metals and among the metals at each trimester.

We used several analytic approaches to study the relationships between metal exposure and risk of HDP. Adjusted Odd ratios (aOR) and their corresponding 95% Confidence intervals (CI) were estimated using logistic regression models to test the relationship between metals and GH overall versus normotensive status or using multinomial regression models with HDP defined according three mutually exclusive groups (normotensive, GH and PE). All metals were individually tested in each model as a continuous as well as a categorical variable (tertiles) to consider different alternatives for modeling exposure. The results were grouped in the tables according to the type of specimen collected (blood versus urine) and the trimester of specimen collection. The p-value (p) <0.05 was considered to indicate statistical significance. In order to control for potential confounding bias, covariates were selected either a priori on the basis of evidence from the literature (maternal age) or according to the change-in-estimate method. All covariates among the list presented above were examined individually and those which changed the OR by +/-10% for the relationship between metals and HDP were considered as confounders and were included in each corresponding multivariate model. In order to increase power in the multivariate models, covariates were considered in a continuous form when possible and missing

data were ignored. This may have reduced the number of women in certain multivariate models but as missing data were rare and considered as “missing completely at random (MCAR)”, this did not have significant impact on the results.

We also investigated the associations between metals and HDP through the potential modification effect of infant sex. We were not able to test the interaction according to maternal smoking status because of the small number of smokers in our study. The results were stratified if the interaction term was significant at p <0.05. In addition we conducted two sensitivity analyses: 1) with fish consumption in the model of mercury and 2) including all blood metals in the same model at each trimester.

In a separate set of analyses, we also explored the relationship between concurrent measures of metals and mean blood pressure using linear generalized estimating equation models, which take into account the clustering within each individual caused by the repeated- measurements design. Models incorporated a first-order autoregressive correlation pattern for the repeated events. The mean arterial pressure was estimated for each woman at each visit (from each individual measure collected in the corresponding visit) and was used as continuously measured outcomes in the linear generalized estimating equations regression models in order to estimate the adjusted beta coefficients (ß) and their corresponding 95%CI. Unlike to HDP, mean blood pressure took into account the measurement of blood pressure in the first trimester. Metals were transformed into their natural-logarithm (ln) to approximate a normal distribution. Linear generalized estimating equation models were developed using concurrent measures of metals and mean arterial pressure at visits 1 and 3. This model allowed the examination of the direct impact of each metal taken at the same time window that blood pressure was measured. It also allowed

examining the association with the blood pressure and not only with the pathologic outcome of hypertension in pregnant women. Since urinary As was measured only once during the first visit, we used linear regression to analyze its association with blood pressure measured at visit 1. All statistical analyses were performed with IBM SPSS Statistics Version 22, SAS 9.4 for Windows, StataSE 14 and R for Windows 3.2.2.

Results

The majority (82%) of the participants were Caucasian, university educated, multiparous, non- smokers, had a household income more than $90,000, and did not consume alcohol in the 3 months before visit 1. The number of women with GH overall was 187 (9.8%) and within this group, 129 (6.8%) had GH without PE, while 58 (3%) had PE. Women with chronic hypertension (n = 58 (3%)) or without data on hypertension status (n = 34 (1.8%)) were treated as missing data with respect to HDP. The study population characteristics are shown in Table 1. The mean maternal age was similar across study groups. The proportion of obesity was significantly higher among women with PE (44.2%) compared with normotensive women (11.6%). Weight gain during pregnancy was also higher in women who developed HDP than among normotensive women. Fish consumption and birth weight were higher in normotensive women. Ethnicity, BMI, education, household income, parity, fish consumption, and birth weight were also significantly associated with HDP.

Spearman correlations between the first and third trimester concentrations of maternal blood metals were significant: Spearman's rho (r) = 0.41 (p <0.001), r = 0.75 (p <0.001), r = 0.64 (p <0.001), r = 0.76 (p <0.001) and r = 0.66 (p <0.001), respectively for As, Pb, Cd, Hg and Mn (Table S1). Similarly first trimester concentrations of blood and urinary As were significantly and

positively correlated for arsenobetaine (r = 0.55, p <0.001), for total urinary As (r = 0.43, p <0.001), for DMA (r = 0.23, p <0.001) and for inorganic As (r = 0.23, p <0.001) (Table S2). Correlations between metals measured during the same trimester were generally low with the highest correlation being between blood As and Hg (r = 0.39) in the third trimester (Tables S3- S5).

In the 1st and 3rd trimesters GM blood Hg concentrations were significantly lower in women who developed GH overall (0.36 µg/l), GH (0.37 µg/l) or PE (0.34 µg/l) compared to women who remained normotensive (0.50 µg/l) (Table 2, Fig. S2). Both GM blood Pb and Mn concentrations at 1st trimester were significantly lower in women who went on to develop HDP than those who remained normotensive women. Maternal concentrations of blood Cd and As were not significantly associated with HDP. The percentage distribution of tertiles of metal concentrations according to HDP is presented in Table S6.

In the multivariate analysis, a significantly lower risk of GH overall (aOR = 0.62; 95% CI 0.39, 0.98) in the 3rd trimester was observed at the 3rd tertile (> 0.82 µg/l) of blood Hg (Table 3). When 3rd trimester blood Hg was examined as a continuous variable, a lower risk of GH overall was observed (aOR = 0.69; 95% CI 0.50, 0.96) (Table S8), even when controlling for other metals in the model (aOR = 0.66; 95% CI 0.47, 0.93) (Table S10). Blood Hg was not significantly associated with blood pressure in the cross-sectional analysis (Table 5).

First trimester blood manganese (> 9.89 µg/l versus <7.69 µg/l) was associated with a lower risk of developing GH overall (aOR = 0.68; 95% CI 0.46, 0.99) and PE (aOR = 0.48; 95% CI 0.23, 0.98) (Table 3) and was still associated with a significantly lower risk when examined as a continuous variable, adjusting for the other metals (Table S10). When stratified by infant sex,

the risk of GH overall for those in the 3rd tertile for manganese was significantly lower in women delivering male infants (aOR = 0.41; 95% CI 0.24, 0.70) than female infants (aOR = 1.27; 95% CI 0.69, 2.34) (Table 4). In contrast, when blood Mn was modeled with concurrent blood pressure, a significant positive association was observed (aß=1.52; 95% CI: 0.80-2.24) (Table 5).

First trimester blood As (> 0.97 versus <0.60 µg/l) was associated with an almost tripling of the risk of PE (OR = 2.75; 95% CI 1.13, 6.73), adjusted for the other 4 metals (Table S10). In the 3rd trimester, a 1-unit increase in ln-blood As was associated with a significant increase in the risk of PE (aOR = 1.16; 95% CI 1.03, 1.30) (Table S8), even after adjusting for other metals (OR = 1.13; 95% CI 1.00, 1.28) (Table S10). The risk of GH overall associated with As was significantly higher among women carrying male fetuses (aOR = 1.85 (95%CI: 1.02, 3.36) for As level 0.60-0.97 µg/l versus < 0.60 µg/l) than those carrying female fetuses (aOR=0.78 (0.44, 1.37)) (Table 4). Concurrent measures of blood As and blood pressure displayed a significant negative association (beta = - 0.59; 95% CI -0.92, -0.26) (Table 5).

Neither urinary DMA, arsenobetaine nor inorganic As were significantly associated with the risk of HDP; however, a significant negative association was observed between concurrent measures of blood pressure and DMA (Table 6). The second tertile of total urinary As was associated with a lower risk of PE (Table 3) (this result may be due to a type I error). No significant associations were observed between blood Pb or Cd concentrations and the risk of HDP, although the risks for 3rd trimester concentrations were above 1.0 for Pb (Table 3). Also the association between Pb, Cd or Hg concentrations and HDP was not modified by infant sex.

Discussion

Mn and HDP

In our study, the GM of Mn concentrations (measured at first trimester) were lower in patients who developed GH overall (8.43 µg/l) than in normotensive women (8.83 µg/l). The highest tertile of Mn (> 9.89 µg/l) was associated with a decrease in risk of GH overall and PE risk. This finding is consistent with a previous case-control study (N = 108) that reported that serum mean Mn concentrations were significantly lower in PE (0.08 µg/l) than in control women (0.14 µg/l) (Sarwar et al. 2013). Similar results were also observed by Al-Jameil et al. (2014) and Maduray et al. (2017). However, our findings contrast with those reporting an association between Mn and the higher risk of HDP. In a cohort study of 364 healthy pregnant women, Vigeh et al. (2013) observed that maternal blood Mn concentrations measured in the first (mean = 18.6 µg/l) and second trimesters (mean = 18.9 µg/l), were associated with a higher risk of GH. Third trimester results were not statistically significant. Similar results were reported by Vigeh et al. (2006). This

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