Scholarly article on topic 'Drinking Water Disinfection By-products, Genetic Polymorphisms, and Birth Outcomes in a European Mother–Child Cohort Study'

Drinking Water Disinfection By-products, Genetic Polymorphisms, and Birth Outcomes in a European Mother–Child Cohort Study Academic research paper on "Environmental engineering"

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Academic research paper on topic "Drinking Water Disinfection By-products, Genetic Polymorphisms, and Birth Outcomes in a European Mother–Child Cohort Study"

Original Article

Drinking Water Disinfection By-products, Genetic Polymorphisms, and Birth Outcomes in a European Mother-Child Cohort Study

Manolis Kogevinas,a'b'c'd Mariona Bustamante,^0^ Esther Gracia-Lavedán,a'h'° Ferran BaUester,cfg Sylvaine Cordierh Nathalie Costeth Ana Espinosa,a'h'c Regina Grazuleviciene,j Asta Danileviciute,j Jesus Ibarluzea,°'k'1 Maria Karadanelli,m Stuart Krasner,n Evridiki Patelarou,0 Euripides Stephanou,p Adonina Tardón,cq Mireille B. Toledano,r John Wright,s Cristina M. Villanueva,a'h'°

and Mark Nieuwenhuijsena'h'°

Background: We examined the association between exposure during pregnancy to trihalomethanes, the most common water disinfection by-products, and birth outcomes in a European cohort study (Health Impacts of Long-Term Exposure to Disinfection By-Products in Drinking Water). We took into account exposure through different water uses, measures of water toxicity, and genetic susceptibility. Methods: We enrolled 14,005 mothers (2002-2010) and their children from France, Greece, Lithuania, Spain, and the UK. Information on

Submitted 25 August 2015; accepted 25 July 2016.

From the aBarcelona Institute for Global Health (ISGlobal), Barcelona, Spain; bUniversitat Pompeu Fabra (UPF), Barcelona, Spain; cCIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain; ''Municipal Institute of Medical Research (IMIM-Hospital del Mar), Barcelona, Spain; eCenter for Genomic Regulation (CRG), Barcelona, Spain; fFISABIO-UJI-University of Valencia Unit of Research, Valencia, Spain; gCIBERESP, Madrid, Spain; hINSERM (National Institute of Health and Medical Research) U1085-IRSET, Rennes, France; University of Rennes

I, Rennes, France; ^Department of Environmental Sciences, Vytauto Did-ziojo Universitetas, Kaunas, Lithuania; kBiodonostia Health Institute, San Sebastián, Spain; 'Public Health Department of Gipuzkoa, San Sebastian, Spain; mDepartment of Marine Sciences, University of the Aegean, Mytilene, Greece; "Metropolitan Water District of Southern California, La Verne, CA; "Florence Nightingale Faculty of Nursing and Midwifery, King's College London, London, United Kingdom; pEnvironmental Chemical Processes Laboratory (ECPL), Department of Chemistry, University of Crete, Heraklion, Greece; qIUOPA, Universidad de Oviedo, Asturias, Spain; rMRC-PHE Centre for Environment and Health, School of Public Health, Faculty of Medicine, Imperial College London, London United Kingdom; and sBradford Institute for Health Research, Bradford Royal Infirmary, Bradford, United Kingdom.

The HIWATE project was funded under the EU Sixth Framework Program for Research and Technological Development by the Research Directorate-Biotechnology, Agriculture and Food Research Unit (Contract No. Food-CT-2006-036224). The Joint Environment & Human Health Programme [NERC UK Grant NE/E008844/1].

The authors report no conflicts of interest.

IqqqI Supplemental digital content is available through direct URL citations

-in the HTML and PDF versions of this article (www.epidem.com).

Correspondence: Manolis Kogevinas, Barcelona Institute for Global Health (ISGlobal), 88 Dr Aiguader Rd, Barcelona 08003, Spain. E-mail: manolis. kogevinas@isglobal.org.

Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

ISSN: 1044-3983/16/2706-0903

DOI: 10.1097/EDE.0000000000000544

Epidemiology • Volume 27, Number 6, November 2016

lifestyle- and water-related activities was recorded. We ascertained residential concentrations of trihalomethanes through regulatory records and ad hoc sampling campaigns and estimated route-specific trihalo-methane uptake by trimester and for whole pregnancy. We examined single nucleotide polymorphisms and copy number variants in disinfection by-product metabolizing genes in nested case-control studies. Results: Average levels of trihalomethanes ranged from around 10 ^g/L to above the regulatory limits in the EU of 100 ^g/L between centers. There was no association between birth weight and total trihalomethane exposure during pregnancy (S = 2.2 g in birth weight per 10 ^g/L of trihalomethane, 95% confidence interval = 3.3, 7.6). Birth weight was not associated with exposure through different routes or with specific trihalomethane species. Exposure to trihalomethanes was not associated with low birth weight (odds ratio [OR] per 10 ^g/L = 1.02, 95% confidence interval = 0.95, 1.10), small-for-gestational age (OR = 0.99, 0.94, 1.03) and preterm births (OR = 0.98, 0.9, 1.05). We found no gene-environment interactions for mother or child polymorphisms in relation to preterm birth or small-for-gestational age. Conclusions: In this large European study, we found no association between birth outcomes and trihalomethane exposures during pregnancy in the total population or in potentially genetically susceptible subgroups. (See video abstract at http://links.lww.com/EDE/B104.)

(Epidemiology 2016;27: 903-911)

Disinfection by-products are formed as a side reaction of water disinfection. Chlorinated water contains hundreds of disinfection by-products of which trihalomethanes and haloace-tic acids are the most common compounds. concern about the potential health risks of exposure to disinfection by-products has focused on cancer1 and birth outcomes.2,3 In animal studies, high doses of chloroform (cHcl3) and various haloacetic acids and haloacetonitriles have been associated with mental growth retardation.4 At least 20 studies of different design and quality of information have examined the relation between fetal growth and disinfection by-products in humans. A recent meta-analysis found a 1% increased risk for small-for-gestational age

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(SGA) but no evidence for associations between third trimester trihalomethane exposure and low birth weight (LBW), term LBW, preterm births.5 Only two studies have examined whether genetic variation may affect risk of birth outcomes associated with exposure to trihalomethanes.6,7

Exposure assessment has been a major limitation of most studies that have used predominantly ecologic estimates of trihalomethane exposures in water supply zones. Some studies have combined individual information on water use with water zone estimates but fewer have examined different routes of expo-sure.2,3,6,8-11 Exposure to trihalomethanes and other volatile disinfection by-products occurs predominantly through inhalation and absorption, during activities, such as showering, bathing, and swimming.12,13 For nonvolatile disinfection by-products, such as haloacetic acids, ingestion is the main route of expo-sure.14 Epidemiologic studies have used trihalomethanes as a proxy for total disinfection by-product exposure, which may underestimate exposure to disinfection by-products.15,16

Analyses of the association of trihalomethanes with birth outcomes for country-specific populations included in Health Impacts of Long-Term Exposure to Disinfection By-Products in Drinking Water (HiWate) have been published previously.8-10,17,18 In this article, we report the results of the analysis of pregnancy outcomes in five pooled mother-child cohorts from France, Greece, Lithuania, Spain, and the UK. We examined exposure to trihalomethanes through drinking, bathing, and showering activities applying the same protocol for exposure assessment and also present associations in potentially genetically susceptible groups in nested case-control studies.

METHODS

Study Population

This European mother-child cohort study was conducted within the European HiWate project19 and includes five cohorts (Table 1). We enrolled 14,005 mother-child pairs in 2002-2010 in obstetric units located in parts of five European countries: Greece (Rhea study, Heraklion [Crete]); Spain (INMA-Infancia y Medio Ambiente Project, Sabadell [Catalonia], Valencia, Asturias, Gipuzkoa [Basque Country]); United Kingdom (Born in Bradford study (BiB), Bradford); France (Pelagie study, Brittany); and Lithuania (Kaunas).

Detailed information regarding exposure and outcome of interest was collected from face-to-face interviews or self-reports during pregnancy together with birth records. Questionnaires were used in the five studies to collect information on sociode-mographic, lifestyle, nutrition, occupation, medical, and reproductive history, family history, and environmental exposures. Information was available in all studies on the main a priori risk factors for birth outcomes, including maternal age and education, socioeconomic status, parity, smoking, and alcohol consumption.

Birth Outcomes

Birth weight in grams, head circumference in cm, gesta-tional age (weeks), gender, and mode of delivery were extracted

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from birth records, where available. Birth weight was analyzed as a continuous outcome, and was dichotomized as LBW, defined as weight less than 2,500 g, and term LBW, defined as a birth weight below 2,500 g after at least 37 completed weeks of gestation.

Newborns SGA were defined as those who weighed less than the 10th percentile of the cohort-specific reference of fetal growth, stratified by week of gestation and gender. In two studies (BiB and RHEA), we used customized (internal) models on fetal growth restriction.

Gestational age was based on last menstrual period and/ or ultrasound-based estimated date of conception. Analyses related to gestational duration considered both the continuous outcome and dichotomized values of gestational duration (prematurity) defined as gestation length <37 completed ges-tational weeks. Mode of delivery was grouped into vaginal deliveries and Caesarean sections.

Exposure Assessment for Water Contaminants

All studies had information on water intake and sources of drinking water, information on showering, bathing, and swimming pool use during pregnancy. Information from questionnaires was harmonized before the analysis. The evaluation of trihalomethane concentrations in drinking water in the study areas was done through the use of routinely collected trihalo-methane data for regulatory purposes and was enhanced with information from disinfection by-product samples collected and measured within the HiWate project.20 A description of trihalomethane levels available in each center is provided in eAppendix1 (http://links.lww.com/EDE/B93).

Trihalomethane data were modeled based on available water quality parameters, treatment, and water source for the study regions. A separate model was built in each country (region in the case of Spain) for total trihalomethanes, chloroform, and total brominated trihalomethanes following similar methods as discussed in study-specific published papers.8-10,17,18 Linear regression and generalized additive models were fitted using geographical and temporal variables (month, year). Among models retaining significant variables (P < 0.05), criteria to select the final model included the adjusted R2 and the Akaike Information Criteria. General additive models were used to fit a smooth function of level by month that was used to predict levels for months without observations. Final models predicted a monthly level of trihalo-methanes from conception until delivery in all study subjects.

Uptake of trihalomethanes (total dose log transformed to normalize) was estimated using a combination of modeled trihalomethanes, information on personal activities via ingestion, showering, and bathing, and uptake factors based on the literature21-23 and in an earlier analysis in Spain.9 Uptake factors used in the analysis for water ingestion were 0.0049 ^g/^g/L for total trihalomethanes, 0.00490196 ^g/^g/L for chloroform and 0.00111848 ^g/^g/L for brominated trihalomethanes; for showers, the corresponding values were 0.001321, 0.00153626, and 0.00135206 ^g/min/^g/L; for baths, the corresponding values were 0.001538, 0.00132075, and 0.00129571 ^g/min/^g/L.

© 2016 Wolters Kluwer Health, Inc. All rights reserved.

TABLE 1. Subjects, Birth Outcomes, Lifestyle, and Water-related Variables in the European Study Population and by Cohort3

Greece Spain France Lithuania UK

All (Heraklion) (Four Areas) (Britanny) (Kaunas) (Bradford)

14,005 1,359 (10%) 2,473 (18%) 3,322 (24%) 4,158 (30%) 2,693 (19%)

Sex of newborn

Female 6,835 (49%) 670 (49%) 1,200 (49%) 1,640 (49%) 2,030 (49%) 1,295 (48%)

Male 7,164 (51%) 689 (51%) 1,273 (52%) 1,681 (51%) 2,128 (51%) 1,393 (52%)

Low birth weight 704 (5%) 73 (6%) 125 (5%) 108 (3%) 202 (5%) 196 (7%)

Term low birth weight 295 (2%) 25 (2%) 68 (3%) 39 (1%) 72 (2%) 91 (4%)

Small for gestational 1,302 (10%) 102 (9%) 240 (10%) 332 (10%) 412 (10%) 216 (9%)

age Gestational age 39.3 (1.7) 38.2 (1.6) 39.6 (1.7) 39.4 (1.5) 39.2 (1.7) 39.5 (1.9)

(weeks)

Preterm births 791 (6%) 154 (12%) 113 (5%) 126 (4%) 233 (6%) 165 (6%)

BMI mother (kg/m2)

<20 2,392 (18%) 192 (15%) 403 (16%) 854 (26%) 672 (16%) 271 (11%)

20-25 6,664 (49%) 664 (53%) 1,421 (58%) 1,867 (57%) 1,749 (42%) 963 (38%)

25-30 3,057 (22%) 244 (20%) 456 (18%) 413 (13%) 1,207 (29%) 737 (29%)

>30 1,575 (12%) 143 (12%) 192 (8%) 163 (5%) 530 (13%) 547 (22%)

Education of mother

Low 1,614 (12%) 265 (21%) 605 (25%) 23 (1%) 244 (6%) 477 (18%)

Medium 6,016 (43%) 632 (50%) 1,018 (41%) 1,225 (37%) 1,719 (41%) 1,422 (53%)

High 6,043 (44%) 358 (29%) 845 (34%) 2,066 (62%) 2,195 (53%) 579 (22%)

Other 184 (1%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 184 (7%)

Parity

Nulliparous 6,438 (46%) 484 (38%) 1,390 (56%) 1,473 (45%) 2,032 (49%) 1,059 (40%)

1+ 7,425 (54%) 798 (62%) 1,081 (44%) 1,837 (56%) 2,126 (51%) 1,583 (60%)

Smoking during pregnancy, mother

Never 10,150 (84%) 881 (76%) 1,969 (82%) 1,217 (71%) 3,849 (93%) 2,234 (83%)

Ever 1,986 (16%) 280 (24%) 438 (18%) 500 (29%) 309 (7%) 459 (17%)

Secondhand smoke, mother

No 5,822 (49%) 86 (7%) 900 (38%) 876 (61%) 2,110 (51%) 1,850 (69%)

Yes 6,058 (51%) 1,183 (93%) 1,499 (63%) 553 (39%) 2,000 (49%) 823 (31%)

Numbers and percentages or mean and standard deviation (SD), HiWate cohort (n= 14,005). aTotals may not add up because of missing values. BMI indicates body mass index.

Because a bromoform uptake factor was only available for showering, the average of bromodichloromethane and dibro-mochloromethane uptake factors were used for the three brominated trihalomethanes. Swimming pool uptake factors and calculation of uptake from swimming were available only in the Spanish INMA cohort9 and are not reported. A 90% reduction in ingestion was applied if a home filter was used; no information was available for factors that could affect efficiency of trihalomethane removal such as frequency at which filters were changed. Average trihalomethane uptake over the whole pregnancy was calculated, as well as in the first, second, and third trimesters separately to allow for evaluation of critical windows of exposure. Bathing and showering uptakes were added, and total household uptake was calculated by adding ingestion, showering, and bathing.

© 2016 Walters Kluwer Health, Inc. All rights reserved.

Genetic Nested Case-Control Study

We designed nested case-control studies on preterm births and a combination of SGA to evaluate the potential influence of genetic polymorphisms in connection to exposure to trihalomethanes. Controls were selected from the cohorts and were matched to cases on ethnic group or country of origin and sex, and were not preterm, not SGA, and not large for gestational age. The same set of controls was used for the analysis of both phenotypes. A total of 2,159 DNA samples were included initially in the study and of those 1,908 were finally included in the analysis. There were 964 maternal samples (348 SGA, 251 preterm, and 395 controls) and 944 child DNAs (349 SGA, 218 preterm, and 400 controls); these numbers do not add because a small proportion of children were included in both the SGA and the preterm analysis. Maternal blood was not available in the Pelagie (France) cohort.

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Genotyping was not available for BiB at the time of this analysis. DNA extraction methods can be found elsewhere.24 DNA was quantified using the PicoGreen dsDNA kit (Invitrogen, Thermo Fisher Scientific Inc.) and normalized to 40-60 ng/pl.

Candidate genes that are known to participate in disinfection by-product detoxification were examined, including CYP1A locus (CYP1A1 and CYP1A2), CYP2A6, CYP2D6, CYP3A locus (CYP3A4, CYP3A5, CYP3A7, and CYP3A43), CYP2E1, GSTZ1, and GSTT locus (GSTT1, GSTT2, and GSTT2B). Genetic variants in detoxifying genes GSTM and GSTA loci were also explored. A detailed description of the selection of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs), genotyping procedures and quality control are presented in the eSupplement on methods and in eTable 1 (http://links.lww.com/EDE.B94).

Statistical Analysis

We evaluated the association between birth weight and average residential trihalomethane levels by linear regression, adjusting for gestational age and other potential confounders. Trihalomethane exposures were examined in three groups: total trihalomethanes, chloroform, and brominated trihalomethanes. Adjustment for potential confounders was predefined based on prior knowledge on potential confounders with two levels of adjustment applied. A basic model included variables expected to be available in all subjects of all participating studies and that had been used in most previous studies: center (and in Spain, also area, Gipuzkoa [Basque Country], Sabadell, Asturias, and Valencia), infant sex, gestational age linear and quadratic term, mother's ethnicity, and parity. We included infant sex in the models to be compatible with previous studies although infant sex should not be considered a typical confounder; results changed minimally (second decimal) when not adjusting for sex (not shown). Further adjusted models included, in addition, maternal age, maternal height, maternal prepregnancy weight, maternal education, and maternal smoking during pregnancy; results for these models were similar to those of the basic adjustment models and are not shown. Logarithmic transformation of uptake of trihalomethanes was used in corresponding models. We used logistic regression to analyze dichotomous outcomes adjusting for potential confounders and general additive models to evaluate the shape of the dose-response curve. A meta-analysis was performed to take into account the heterogeneity between the cohorts. A random effects model was used and a heterogeneity test based on the Q statistic was performed considering a P value below 0.10 as statistically significant.

We assessed gene-environment (G * E) interactions in the nested case-control study for total trihalomethanes only using unconditional logistic regression, adjusting for infant sex, ethnicity, parity, smoking during pregnancy, and cohort. Dominant genetic models were tested. To take into account the heterogeneous distribution of both disinfection byproducts (DBPs) and allele frequencies of selected SNPs in the cohorts, we included cohort SNP and cohort-total trihalomethane

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interaction terms in the models. Only those associations with consistent results between these statistical models were reported as significant results. To control for multiple testing, we applied Bonferroni correction for the 43 independent associations examined (P value 0.0011).

Ethics

The protocols of all studies were approved by local ethics committees. All subjects signed a consent form that includes the use of genetic data. Standard procedures for the protection of confidential individual information have been applied. Information that might identify a specific individual was never released or transferred between participating centers.

RESULTS

Total trihalomethane levels in the water differed considerably between the regions of the European countries studied depending on water source and type of treatment (Figure). Differences were also observed within countries. The highest levels (average total trihalomethanes above the regulatory limit in the EU of 100 pg/L) were observed in parts of Spain (Sabadell), levels around 50 pg/L were observed in France and the UK, while the lowest levels (around 10 pg/L) were observed in some parts of Spain and in Lithuania and Greece. The distribution of the individual trihalomethanes also differed between the parts of the countries studied with proportionally high levels of brominated compounds found in two of the regions in Spain studied (Sabadell, Valencia) and France compared with the UK or the Basque Country where the main exposure was to chloroform. Between the two low-level regions in the two other countries, Heraklion (Greece) had almost exclusive exposure to brominated compounds, whereas Kaunas (Lithuania) was dominated by chlorinated trihalomethanes.

The overall mean birth weight was 3,333 g/kg (SD: 521) and differed between cohorts (Greece: 3,179 [457]; Spain: 3,256 [478]; France: 3,391 [493]; Lithuania: 3,447 [522]; UK: 3,229 [567]). A high proportion of preterm births was observed in the Rhea cohort in Crete (11.6%). There were wide differences in the level of education and in smoking between cohorts in the regions studied (Table 1).

Water use and other water activities differed by region studied (Table 2). Tap water consumption was highest in Bradford (UK; 83%) and lowest in Heraklion (Greece; 18%). Baths were less frequent in the two regions studied in southern Europe. Swimming pool use varied, with very low use in Heraklion (Greece; 2%) and higher use in Brittany (France) and Spain.

Main Associations with Trihalomethane Exposure

There was no association between birth weight and total trihalomethane exposure during pregnancy (Table 3). The change in birth weight in grams per 10 pg/L increase in total trihalomethanes was 2.2 g (95% confidence interval [CI] = -3.3, 7.6) for the total cohort. The corresponding в coefficients and 95% CI for the individual cohorts were 2.7 (-9.9, 15.3) for Pelagie in Brittany

© 2016 Wolters Kluwer Health, Inc. All rights reserved.

Spain-Sabadell - —HI—• Spain-Asturias---HF

Spain-Gipuzkoa - -ID-*

Spain-Valencia - H

France " "CD-

UK- -ID—

^ Total THMs E-3 Chloroform ^ Brominated THMs

Lithuania - I I l~ 0-

Greece " H

ALL- CI

50 100

FIGURE. Distribution of total trihalomethanes (THMs), CHCl3, and total brominated THM concentrations at the residence during the whole pregnancy in the study population in the regions of the five countries studied, HiWate study.

(France), 73.8 (-17.9, 165) for Rhea in Heraklion (Greece), 8.3 (-4.9, 21.5) for Kaunas in Lithuania, -0.2 (-6.8, 6.4) for INMA in Spain, and -27.5 (-70.2, -15.2) for BiB in the UK. Differences in estimates between countries were examined through a random effects meta-analysis and were not statistically heterogeneous (x2: 5.45, df = 4, P = 0.244). We observed no association for exposure to chloroform or brominated trihalomethanes nor any indication of a dose-response relationship evaluated through the use of quartiles of trihalomethane exposure (Table 3) or the use of splines (not shown). There was no indication of differences in risk by trimester of exposure for total trihalomethanes (Table 3) or for specific trihalomethanes (not shown).

There was no association of birth weight change with uptake of trihalomethanes. The change in birth weight in grams (and 95% CI) for a 10% increase in total trihalo-methanes, chloroform, and total brominated concentrations through ingestion, shower/bath, or total uptake during the whole pregnancy adjusted for potential confounders are shown in Table 3. There were marked differences in uptake of tri-halomethanes through ingestion, showers, and baths between cohorts, reflecting mostly the differences in trihalomethane concentrations in water in each region (country) studied. The total uptake is defined mostly (around 90%) by uptake from showers and baths.

We found no association with exposure to trihalomethanes per 10 Mg/L increase for term LBW (odds ratio [OR] = 1.04, 95%

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CI = 0.96, 1.14), SGA (OR = 0.99, 95% CI = 0.94, 1.03), and preterm births (OR = 0.98, 95% CI = 0.9, 1.05; Table 4). Similarly, risks were close to null when examining separately exposure to chloroform or brominated compounds. No pattern was seen for a dose response when examining quartiles of exposure or using generalized additive models and these birth outcomes for total trihalomethanes or for chloroform or brominated compounds (not shown). Uptake through any route or total uptake was also not associated with any of these outcomes (not shown).

Nested Case-Control Studies on Gene-Environment Interactions

We examined gene-environment interactions for maternal and child genotypes in relation to preterm births and SGA for total trihalomethanes (Table 5). Two genetic variants of the mother (rs743535 in CYP2E1 and GSTT1 CNV) modified the effect of trihalomethane levels on SGA with a P value for the interaction of less than 0.05. In particular, among those mothers bearing GSTT1 null genotype, an increased risk was observed for SGA for a 10 Mg/L increase in total trihalomethanes (OR = 1.4, 95% CI = 0.9, 2.1). None of these interactions persisted after Bonferroni correction for multiple comparisons. Detailed results both on the main genetic effects and on gene-environment interactions on birth outcomes are shown for preterm births in eTable 2 and for SGA in eTable 3 (http:// links.lww.com/EDE.B94).

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TABLE 2. Tap Water Consumption and Other Water Activities by Region3

Greece Spain France Lithuania UK

All (Heraklion) (Four Areas) (Brittany) (Kaunas) (Bradford)

Number of subjects 14,005 1,359 2,473 3,322 4,158 2,693

Tap water

Yes 7,732 (56%) 234 (18%) 1,166 (47%) 1,964 (60%) 2,138 (51%) 2,230 (83%)

No 6,158 (44%) 1,051 (82%) 1,307 (53%) 1,324 (40%) 2,020 (49%) 456 (17%)

Average L/day (if yes) 0.83 1.16 0.96 0.29 0.79 1.23

Bottled wate^5

Yes (main source) 7,311 (69%) 972 (76%) 2,172 (88%) 2,904 (88%) 3,249 (78%) 918 (34%)

No 3,291 (31%) 313 (24%) 301 (12%) 286 (12%) 909 (22%) 1,768 (66%)

L/day (if yes) 1.08 1.21 1.04 1.09 1.09 1.02

Showers

Yes 10,610 (76%) 1,185 (87%) 2,340 (95%) 1,455 (99%) 3,903 (94%) 1,727 (64%)

Min/day 11.3 11.1 9.97 8.2 13.0 12

Yes 4,292 (31%) 71 (5%) 277 (11%) 564 (38%) 1,660 (40%) 1,720 (64%)

Min/day 10.9 12.2 8.95 5.8 8.0 15.7

Only bath

Yes 981 (7%) 17 (1%) 47 (2%) 33 (1%) 166 (4%) 718 (27%)

Min/day 17.7 20.6 19.2 11.9 13.7 18.7

Swimming pools

Yes 2,223 (18%) 25 (2%) 1,046 (43%) 603 (32%) 346 (8%) 203 (8%)

No 10,136 (82%) 1,228 (98%) 1,364 (57%) 1,258 (68%) 3,814 (92%) 2,472 (92%)

aNumbers may not add up because of missing values.

information on bottled water, showers, and baths is available only for a subsample in the Pelagie cohort (France).

DISCUSSION

In this large European study, exposure to trihalomethanes during pregnancy was not associated with birth weight, SGA, LBW, or preterm birth despite the high concentrations of trihalomethanes and/or bromine-containing species in some areas. Results were consistent between European regions studied. We found little evidence of the potential modification of the effect of exposure to DBPs by genetic susceptibility.

The epidemiologic evidence evaluating associations between trihalomethane exposure during pregnancy and fetal growth is extensive. Different methodologies, particularly in exposure assessment, and different exposures and characteristics of the study populations hamper comparisons between studies. Experimental evidence suggests that triha-lomethanes have a harmful effect on fetal growth, but this has not been confirmed in epidemiologic studies. Our results support other recent studies that have applied robust exposure assessments of disinfection by-products.2,3 Our results were consistent across centers, particularly for residential trihalomethane uptake, providing robust combined risk estimates. There is no evidence for an association between preterm delivery and trihalomethane exposure during pregnancy, with most of the studies finding either a null associa-tion25,26 or a protective effect.2,27 The lack of any association in our study is thus consistent with the conclusions of a recent meta-analysis.5

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There is limited evidence on the potential effect modification by genetic variants of key genes for disinfection by-product detoxification in relation to adverse reproductive outcomes. In this European study, we have evaluated an extensive list of genes known to participate in disinfection by-product detoxification, exploring common polymorphisms by using tag SNPs and CNVs. The two interactions reported here, which did not remain significant after multiple testing comparisons, account for maternal genetic variants in CYP2E1 and in GSTT1 genes, two of the most relevant genes for DBP detoxification. The lack of association between these same variants and SGA in the offspring might reflect time and tissue specificity expression of the detoxification genes. In contrast to what has been seen for bladder cancer,28 we found that total trihalomethanes were associated with increased SGA risk among children whose mothers had no copies of a GSTT1 CNV Our results are in line with those reported by Kogevinas et al.,29 where exposure to total trihalomethanes had a mutagenic effect in lymphocytes (increased the number of micronuclei) among GSTT1 null subjects, but not in subjects having at least one copy of the GSTT1 gene. GSTT1 gene lies in a complex genomic region rich in CNVs and encompassing three highly similar genes: GSTT1, GSTT2, and GSTT2B30

A strength of our study is the prospective cohort design, with individual follow-up of pregnancies to determine reproductive outcomes. Most mothers in the European study self reported the last menstrual period during the first trimester of pregnancy

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TABLE 3. Beta Coefficients Showing Estimated Change in Birth Weight in Grams for a 10 p,g/L Increase in Total trihalomethanes (THMs), CHCl3, and Total Brominated Trihalomethane Levels in Drinking Water During Whole Pregnancy and by Trimester, and for a 10% Increase in Uptake of the Same Compounds Through Different Routes3

Beta Coefficient N (95% CI)

THMs in drinking water, whole pregnancy

Total THMs 13,098 2.17 (-3.3,7.6)

Chloroform 13,098 0.97 (-9.5, 11.4)

Brominated 13,098 2.54 (-4.7, 9.7)

Quartiles of total THM in drinking water, whole pregnancy

Quartile 1 (<5.2 ^g/L) 3,261 Reference

Quartile 2 (5.2-24.22 ^g/L) 3,301 22.04 (0.5, 43.5)

Quartile 3 (24.24-47.4 ^g/L) 3,279 20.73 (- -22.1, 63.6)

Quartile 4 (>47.4 ^g/L) 3,257 16.94 (- 24.8, 58.6)

Total THMs in drinking water by pregnancy trimester

First trimester 13,098 1.05 (- 3.7, 5.8)

Second trimester 13,098 1.49 (- 3.3, 6.3)

Third trimester 13,090 2.75 (- 2.2, 7.7)

Uptake total THMs, whole pregnancy

Ingestion 11,036 0.04 (- 0.26, 0.35)

Shower, baths 11,036 0.34 (- 0.28, 0.96)

Total uptake 11,036 0.35 (- 0.28, 0.98)

Uptake chloroform, whole pregnancy

Ingestion 11,036 0.02 (- 0.26, 0.3)

Shower, baths 11,036 -0.03 (- 0.55, 0.49)

Total uptake 11,036 -0.03 (- 0.55, 0.5)

Uptake brominated THMs, whole pregnancy

Ingestion 11,036 0.06 (- -0.17, 0.29)

Shower, baths 11,036 0.42 (- -0.26, 1.09)

Total uptake 11,036 0.43 (- -0.25, 1.12)

aBeta coefficient (95% CI) from linear regression, adjusted for infant sex, gestational age linear and quadratic term, mother ethnicity, parity, and cohort.

when recall is more accurate, and we were able to correct with ultrasound dating. All of the cohorts had detailed individual-level information available on potential confounders/risk factors, which we were able to adjust for in the analyses. This is one of the few studies with detailed water and water-based fluids consumption and report of personal water-related activities during pregnancy. Similar to all the key recent studies, we were able to comprehensively evaluate uptake of trihalomethanes by routes of exposure: ingestion, inhalation, and dermal exposure. The between-country comparisons are informative because cultural differences are related to differential use of water-related activities. Assessment of exposure from showers and baths is particularly important because in several countries exposure to trihalomethanes through ingestion was very low due to the high consumption of bottled water by pregnant women (e.g., Spain and the regions in Greece and Lithuania studied).

We have based our exposure assessment on trihalo-methanes, which are the most prevalent group of disinfection

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TABLE 4. Odds Ratio (95% CI) of Term Low Birth Weight, Small-for-gestational Age, and Preterm Births for a 10 p,g/L Increase of Total trihalomethanes (THMs), Chloroform, and Total Brominated THMs During Whole Pregnancy Adjusted for Potential Confounders

N OR (95% CI)

Term low birth weighta

Total THMs 12,352 1.04 (0.96, 1.14)

chloroform 12,352 1.09 (0.92, 1.3)

Brominated THMs 12,352 1.04 (0.94, 1.16)

Small-for-gestational ageb

Total THMs 12,646 0.99 (0.94, 1.03)

chloroform 12,646 0.98 (0.89, 1.07)

Brominated THMs 12,646 1 (0.94, 1.06)

Preterm birthsc

Total THMs 13,098 0.98 (0.9, 1.05)

chloroform 13,098 0.91 (0.79, 1.05)

Brominated THMs 13,098 0.98 (0.89, 1.08)

aTerm low birth weight: infant sex, gestational age linear and quadratic term, mother's ethnicity, parity, and cohort.

bSmall-for-gestational age: mother's ethnicity, parity, and cohort. cPreterm: birth weight, infant sex, mother's ethnicity, parity, and cohort.

by-products, using thousands of measurements of these compounds in the study areas. Even though most measurements of trihalomethanes took place simultaneously with the development of the cohorts, there is, undoubtedly, (nondifferential) misclas-sification regarding the evaluation of individual trihalomethane exposures. What is probably more important is misclassification regarding the evaluation of water toxicity when using trihalo-methanes as the basis for the evaluation of the toxicity. Information on other major contaminants, particularly the haloacetic acids, was not available for all centers. Similar trihalomethane concentrations in different regions/countries could potentially mask different mixtures of disinfection by-products with different toxicities. We analyzed chloroform separately from the brominated trihalomethanes that were more prevalent in some areas (e.g., two regions in Spain and the regions studied in France and Greece) as compared with Lithuania and the UK sites and one of the Spanish regions. This is important, as bromine-containing disinfection by-products are of higher health concern. Moreover, the presence of high levels of bromine-containing trihalometh-anes will mean there are high levels of other bromine-containing disinfection by-products,31 some of which are much more toxic than the regulated trihalomethanes.32,33 New methods for evaluating water toxicity, requiring prospective collection of water samples will be needed in future epidemiologic studies to cover this complex pattern of water toxicity.

Overall, in this study of around 14,000 mother-child pairs in eight regions in five European countries with a variation in average levels of total and specific trihalomethanes, we did not find substantial associations between exposure to tri-halomethanes during pregnancy and reproductive outcomes. Although a large part of the population included subjects with

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TABLE 5. Effect of an Increase in Exposure by 10 цд/L Total trihalomethanes on the Association with SGA, Stratified by Maternal Genotype (Genetic Dominant Model)

SNP/CNV

Genotype

Controls

OR (95% CI)

P Value Interaction

CYP2E1

rs743535

GSTT1 CNV

-/-/+, +/+

342 (87.0) 51 (13.0) 69 (18.3) 308 (81.7)

286 (82.7) 60 (17.3) 68 (20.2) 269 (79.8)

1.1 (1.0, 1.2) 0.9 (0.6, 1.1) 1.4 (0.9, 2.1) 1.0 (0.9, 1.1)

low-level exposures, a considerable proportion was exposed to concentrations around 50 ^g/L that have been associated with a 50% increase in bladder cancer risk,1,34 while exposures measured by one center were above the US and EU regulatory limits (80 and 100 ng/L, respectively). The evaluation of genetically susceptible individuals indicated the possibility of gene-environment interactions for genes known to be associated with the metabolism of specific DBPs, such as GSTT1 or GSTZ1, but none of these interactions remained after correcting for multiple comparisons.

In conclusion, results from the HiWate study, together with those of a recent meta-analysis, indicate the lack of any important association of trihalomethanes with a number of adverse birth outcomes.

ACKNOWLEDGMENTS

We would like to thank Emma Goslan in Cranfield University, UK, for her participation in exposure assessment in the HiWate project.

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