Dioxin-like activity and maternal thyroid hormone levels in second trimester maternal serum

American Journal of Obstetrics and Gynecology (2005) 193, 1900–7

www.ajog.org

Dioxin-like activity and maternal thyroid hormone levels in second trimester maternal serum Warren G. Foster, PhD,a,* Alison C. Holloway, PhD,a Claude L. Hughes Jr, MD, PhDb,c Reproductive Biology Division, Department of Obstetrics and Gynecology, McMaster University, Health Sciences Centre,a Hamilton, Ontario, Canada; Department of Medical and Scientific Services, Quintiles Inc,b Research Triangle Park, NC; Department of Biology, East Carolina University,c Greenville, NC Received for publication November 10, 2004; revised June 30, 2005; accepted August 8, 2005

KEY WORDS Dioxins Polychlorinated biphenyls Chemically activated luciferase expression Pregnancy Thyroid

Objective: Developmental exposure to dioxin-like compounds has been associated with cognitive and motor impairments in children. These toxicants have been shown to be thyroid toxicants in animal studies. Therefore, the objective of this study was to quantify the overall dioxin-like activity in maternal serum and determine the association between dioxin-like activity and thyroid hormone levels. Study design: Cross-sectional examination of serum from pregnant women (n = 150) attending a prenatal diagnosis clinic between January 2002 and December 2003. Results: Serum dioxin-like activity was measured in 145 of 150 (96.7%) maternal serum samples. The mean (G SEM) serum lipid-adjusted dioxin-like activity was 0.34 G 0.01 pg/g. Multiple regression analysis failed to demonstrate a relationship between maternal serum dioxin-like activity and serum thyroid hormone levels. Conclusion: Dioxin-like activity is quantifiable in an overwhelming majority of second-trimester maternal serum samples but there was no relationship between dioxin-like activity and thyroid hormone levels in our study population. Ó 2005 Mosby, Inc. All rights reserved.

Prenatal exposure to dioxins and compounds with dioxin-like activity, including polychlorinated biphenyls (PCBs), polychlorinated dibenzofurans (PCDFs), and polychlorinated dibenzodioxins (PCDDs), has been

Supported by grants from the New York Community Trust and the American Chemistry Council. * Reprint requests: Warren G. Foster, PhD, HSC-3N52D, Department of Obstetrics and Gynecology, 1200 Main St West, Hamilton, Ontario, Canada L8N 3Z5. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2005 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.08.013

associated with cognitive and motor impairments postnatally.1-9 These neurologic defects include cognitive and psychomotor developmental delays,2 visual recognition memory impairment, lower verbal and pictoral memory,5,9,10 and delays in verbal comprehension.11 Although children are exposed to dioxin and dioxin-like compounds both in utero and during lactation,12 the adverse effects of these compounds on neurodevelopment appear to be linked specifically to exposure during fetal life.6,10,13,14 Although attenuation of the adverse effects of developmental exposure to PCBs and dioxins by

Foster, Holloway, and Hughes breast feeding and a supportive home environment cannot be overlooked,15 measurement of in utero exposure to dioxin-like compounds is critical to our understanding of potential neurotoxic effects of these environmental exposures. Furthermore, it is critical to determine the mechanism(s) underlying the adverse effects of dioxins during fetal development. In human populations, dysregulation of maternal thyroid function has been associated with motor and cognitive disorders in the offspring.14,16 Furthermore, animal studies have demonstrated that dioxins can interfere with thyroid function,17,18 and altered thyroid function in humans exposed to dioxins has been documented.19-21 Moreover, the epidemiologic evidence suggests that adverse effects of dioxins on thyroid function could occur at background exposure levels.20 Because fetal thyroid hormone production and secretion is not well established until after 20 weeks of gestation in humans,16 alterations in maternal thyroid hormone status by environmental toxicants during the first and second trimesters could have significant implications for fetal neurodevelopment. However, to date, there are no studies in human populations that have assessed the interaction between dioxin exposure and maternal thyroid hormone status during this stage of pregnancy. Furthermore, dioxin and dioxin-like compounds (PCBs, PCDFs, and PCDDs) are present in the environment as complex mixtures of congeners that vary in their metabolism and toxicity. Although residue levels of several PCB and dioxin congeners have been documented in human maternal serum, breast milk, umbilical cord blood, and amniotic fluid,5,7,15,22-33 the measurement of PCB-, PCDF-, and PCDD-specific congeners is very expensive and time consuming and does not necessarily reflect the overall activity of these mixtures in biologic samples. Therefore, alternative methods of determining PCB, PCDF, and PCDD exposure have been developed. Several studies have measured dioxin-like activity in biologic media with the use of hepatoma (H4L1.1c4) cells transfected with an aryl hydrocarbon receptor (AhR)-activated firefly enzyme gene for the enzyme luciferase.34-36 This assay exploits the observation that the majority of the toxic effects of PCDD, PCDF, and dioxin-like PCBs are thought to be mediated through activation of the AhR ligand-activated transcription factor.37 The assay determines the total dioxin-like toxic equivalence (TEQ) in biologic samples and has been validated for use in human serum.38,39 We propose that results from this assay will provide a cost-effective measure of the overall dioxin-like activity present in maternal serum and an indication of the overall potential biologic stimulation received by AhRexpressing tissues. Because the prenatal period appears to be the most sensitive developmental window with respect to adverse neurodevelopmental effects of dioxin and dioxin-like compounds, the objective of the present study was (1) to quantify the dioxin-like activity in

1901 second-trimester maternal serum and (2) to determine whether there is a relationship between dioxin-like activity and maternal thyroid hormone status.

Material and methods Study subjects The current study was approved and conducted in accordance with the McMaster University Research Ethics Board. Study participants were recruited from 310 successive pregnant women attending a prenatal diagnosis clinic at McMaster University Medical Centre between January 2002 and December 2003. McMaster University Medical Centre serves a mixed rural and urban population from South Western Ontario. From the initial cohort of 310 women, 150 pregnant women undergoing a second-trimester amniocentesis for advanced maternal age or maternal anxiety agreed to enter the study. Of the patients not included in the study, 43 of 310 (13.9%) met at least 1 exclusion criterion and 21 of 310 (6.8%) refused to participate (Table I). Exclusion criteria included women without a sufficient command of English, or with a history of illicit drug use, daily alcohol use during the pregnancy, any health condition requiring medical intervention (eg, gestational diabetes, preeclampsia), or prior history of endocrine disease, including thyroid disease, were excluded from the study. At entrance into the study all study participants completed informed consent, demographics, and obstetric history questionnaires at the clinic with the assistance of a research nurse. On the day of their amniocentesis, 2 blood samples (10 mL) were collected in the hospital clinic from all study subjects using standardized procedures for serum collection. Briefly, blood was collected in glass vacutainer tubes, samples were allowed to clot overnight at 4(C, serum was aliquoted for thyroid hormone assay, and a second aliquot collected into precleaned glass vials and frozen at ÿ20(C until required for analysis of dioxin-like activity.

Measurement of serum dioxin-like activity Second-trimester maternal serum dioxin-like activity was determined by using the dioxin responsive-chemically activated luciferase expression (DR-CALUX) assay (Biodetection Systems, Amsterdam, The Netherlands). Maternal serum was tested for the presence of dioxin-like compounds with the use of the DR-CALUX assay according to procedures developed at BioDetection Systems in Amsterdam. Briefly, aliquots of 3.0 mL of maternal serum were extracted by using n-hexane:diethyly ether (97:3) and passed through a silica column containing concentrated H2SO4 (33%) to remove acid-labile matrix components. The cleaned extracts were evaporated and redissolved in 8 mL DMSO and dioxin-like activity

1902 Table I

Foster, Holloway, and Hughes Details of patient enrollment into the study

Category

N

Number of patients invited to participate in the study Number of patients meeting inclusion criteria Number of patients consenting to the study

310

Reasons for nonentry into study Meet at least 1 of the exclusion criteria Refused to participate Cancelled amniocentesis procedure Unable to provide timely entry questionnaires Unable to contact the patient Total

Percentage

177

57.1

150

48.4

43

13.9

21 4 45

6.8 1.3 14.5

20 133

6.5 42.9

measured by using rat H4IIE hepatoma (H4L1.1c4) cells. The hepatoma cells were transfected with an AhR-controlled luciferase reporter gene construct (pGudlic1.1) and were grown in a 96 microwell plate. The samples and the TCDD standards were added to triplicate microwells using 0.5% DMSO as a vehicle and incubated for 24 hours. Cells were then washed twice with phosphate-buffered saline, 75 mL of cell lysis reagent was added, and the cells were centrifuged at 2000g for 1 minute. Luciferase activity was measured by pipetting 20 mL of the supernatant into 96-well microtiter plates and adding 100 mL of luciferin assay mix. The solution was mixed thoroughly and measured with a Luminoskan RS Luminometer (GMI, Inc, Ramsey, MN). Nondetectable values were assigned half the detection limit. Serum lipid levels were determined gravimetrically by routine methods by using aliquots of hexane extracts for each sample.

Radioimmunoassays Circulating levels of thyroid-stimulating hormone (TSH) and thyroxine (T4) were measured with the use of commercially available RIA kits (ICN Pharmaceuticals, Costa Mesa, CA). The limit of detection for TSH was 0.04 mIU/mL and the intra-assay coefficient of variation was 5.2% and for T4 the limit of detection was 0.76 mg/dL and the intra-assay coefficient of variation was less than 9%. All samples were analyzed on the same day in a single assay and thus no interassay coefficient of variation was calculated. Patients were grouped into hypothyroid, euthyroid, and hyperthyroid categories, on the basis of clinically significant thyroid hormone levels as defined by the assay kits used for their analyses. The TSH group ranges were as follows: hyperthyroid, 0.00 mIU/mL to 0.29 mIU/mL; euthyroid, 0.30 mIU/mL to 6.50 mIU/mL; and hypothyroid more than 6.50 mIU/mL. The T4 expected

group ranges were as follows: hypothyroid, 0.0 to 4.99 mg/dL; euthyroid, 5.0 mg/dL to 11.49 mg/dL; and hyperthyroid, 11.5 mg/dL to 19.5 mg/dL.

Statistical methods Descriptive statistics were completed for all outcome variables measured. Data were compared by t test and multiple linear regression analysis (SPSS Inc, Chicago IL) to determine the presence and magnitude of between group differences in dioxin-like activity comparing previously defined groups (hypothyroid, euthyroid, and hyperthyroid) on the basis of age, parity, body mass index (BMI), duration of breast feeding in previous pregnancies, and number of cigarettes smoked/day. Data that failed tests for normality and equal variance (serum dioxin-like activity) were normalized by log transformation. Data with a P ! .05 were considered statistically significant.

Results Patient characteristics The mean age of women enrolled in this study was 38.0 G 0.2 years (range 34-44 years) with a median age of 38 years (Table II). The study population was primarily white 130 of 150 (86.7%), Hispanic 6 of 150 (4.0%), or Middle Eastern 9 of 150 (6.0%). Study subjects reported on average 2.9 G 0.1 previous pregnancies with a range of the current pregnancy being their first through to the sixth for 2 women. Three women experienced a failed pregnancy and 17 of 147 (11.6%) women delivered preterm (!37 weeks). Of the study subjects, 87 of 150 (58%) women reported that they had previously breast fed their newborn infants and the duration of breast feeding ranged from 0.5 to 64 months with a mean length of breast feeding of 14.2 G 1.4 months per child. The socioeconomic status of our study population was assessed from education and income levels that revealed that 122 of 150 (81.3%) women had postsecondary school training and 120 of 150 (80%) women had a family income greater than $40,000.

Dioxin-like activity Of the 150 serum samples analyzed, there were only 5 samples for which levels were nondetectable. The mean (GSE) lipid-adjusted serum CALUX-TEQ level was 0.34 G 0.01 pg/g serum lipids, median of 0.27 pg/g, and ranged from 0.15 to 0.73 pg/g serum lipids. The CALUX-TEQ level measured in the second trimester serum samples from women who subsequently had a preterm delivery was nonsignificantly higher than in those with term deliveries (0.35 G 0.04 vs 0.31 G 0.01 pg/g serum lipids, respectively). Multiple regression

Foster, Holloway, and Hughes

1903

Table II Summary characteristics of pregnant women (n = 150) that enrolled in the study to quantify dioxin-like activity in second-trimester maternal serum Characteristic

Mean (G SEM) Median

Age 38.0 G 0.2 BMI 25.1 G 0.4 Parity 2.9 G 0.1 Gestation length (wks) 39.2 G 0.2

38 15.4 3 40.0

Ethnic background Number White 130 Native Canadian Indian 2 African Canadian 1 Hispanic 6 Asian 1 East Indian 1 Middle Eastern 9

Percentage 86.7 1.3 0.7 4.0 0.7 0.7 6.0

Household income !25th percentile 25-75 percentile O75th percentile

Number 30 111 9

Percentage 20 74 6

Education !25th percentile 25-75 percentile O75th percentile

Number 28 96 26

Percentage 18.7 64.0 17.3

Range 34-44 15.0-24.4 1-6 25*-42

* One mother with clinical hyperthyroidism spontaneously aborted at 25 weeks of gestation.

analysis that used patient age (coefficient of regression = ÿ0.002 and P = .32), number of soy food meals (ÿ0.002, .20), number of Great Lakes fish meals (ÿ0.001, .68), BMI (0.006, .57), parity (ÿ0.009, .32), duration of breast feeding (0.009, .72), and number of cigarettes smoked per day (ÿ0.002, .35) failed to reveal any statistically significant relationships. Lifestyle factors such as dietary fat and cigarette smoking are thought to be potential sources of dioxinlike compounds and therefore maternal dietary and smoking habits during pregnancy were determined. None of the study subjects reported that they were vegetarian. Consumption of soy products was used as a surrogate marker for women who ate an Asian-style diet but were not vegetarian, however; the mean number of meals containing soy products was low with just 2.4 G 0.5 meals per month consumed and a range of 0 to 40 meals per month. Great Lakes fish were consumed by just 22 of 150 (14.7%) women in this study, whereas 126 of 150 (84.0%) women reported eating ocean caught fish (Table III). The number of Great Lakes fish meals consumed was low with more meals of ocean caught fish being consumed (0.2 G 0.1 vs 3.1 G 0.2, respectively). Fast food meals were consumed by 146 of 150 (97.3%) study subjects with a mean of 2.4 G 0.1 meals per month and a range of 0 to 5 meals per month. Wild game and fowl was rarely consumed by the study subjects. In this

Table III The number (percentage) of study subjects that reported consuming different diet components and the mean (GSEM) number of meals per month consumed by study subjects during the first trimester of pregnancy along with selfreported cigarette smoking habits Category

Number

Mean (G SEM) Median Range

Great Lakes fish Ocean fish Fast food meals Game and fowl Soy products

22 (14.7) 126 (84) 146 (97.3) 19 (12.7) 62 (41.3)

0.2 G 0.1 3.1 G 0.2 2.4 G 0.1 0.07 G 0.02 2.4 G 0.5

Smoking habits Nonsmoker Daily smoker Occasional smoker Quite smoking

Number 109 25 9 7

Percentage 72.7 16.7 6.0 4.7

0 3.0 2.0 0 0.2

0-3 0-15 0-5 0-2 0-40

population of pregnant women, 109 of 150 (72.7%) women reported that they were nonsmokers, whereas 25 of 150 (16.7%) women reported that they were daily cigarette smokers. The mean number of cigarettes smoked per day was 13.8 G 1.5 with a minimum of 3 and a maximum of 25 cigarettes smoked per day. The median number of cigarettes smoked per day was 12.0. Dioxin-like activity in smokers versus nonsmokers was 0.30 G 0.02 versus 0.31 G 0.02 pg/g, respectively.

Thyroid function Of 150 subjects for which second trimester serum samples were obtained, 131 (87.3%) were euthyroid, 6 (4%) were hypothyroid, 12 (8%) were subclinically hypothyroid, and 1 (0.4%) was hyperthyroid. No statistically significant relationship was found between dioxin-like activity in the serum and circulating levels of TSH or T4 (Figure). Subjects were grouped according to quartiles on the basis of serum CALUX-TEQ level. Although serum CALUX-TEQ results covered a narrow range, with the highest activity level being just 4.9 times greater than the lowest, serum lipid-adjusted dioxin-like activity levels by percentile group were significantly different between the highest and lowest quartile (P = .001). Comparison between quartiles revealed that there are significant differences (P ! .05) in serum dioxin-like activity across the groups. However, no differences were found in circulating TSH and T4 levels when compared across the highest and lowest quartiles.

Comment The objective of the current study was to quantify dioxin-like activity in second-trimester maternal serum of women with background exposure to PCBs, PCDFs, and PCDDs. Dioxin-like activity was quantified in 145

1904

Figure The relationship between maternal second trimester (A) thyroid stimulating hormone and (B) total serum thyroxin (T4) and dioxin-like activity was assessed in pregnant women (n = 150) attending a prenatal diagnosis clinic at McMaster University Medical Centre. Data were compared by multiple regression analysis controlling for maternal age, parity, cigarette smoking, and history of breast feeding.

of 150 (96.7%) of the second-trimester maternal serum samples examined. Although dioxin-like activity was detected in an overwhelming majority of the study subjects, the level of dioxin-like activity detected was low compared with prior studies that used traditional analytical methods5,8,25,32,40,41 and a recent study that used the CALUX assay.36 No relationship between serum dioxin-like activity and maternal age, BMI, number of previous pregnancies, duration of breast feeding with previous children, diet, or number of cigarettes smoked could be demonstrated. Furthermore, there was no relationship between serum dioxin-like activity and circulating thyroid hormone levels. Our results therefore demonstrate that overall dioxin-like activity is quantifiable in the vast majority of pregnant women in our Canadian cohort and suggest that background levels of dioxin-like chemicals present in maternal serum are

Foster, Holloway, and Hughes too low to be associated with changes in maternal thyroid function. In the current study, dioxin-like activity was measured in 96.7% of the study subjects and the CALUXTEQ values ranged from 0.15 to 0.73 pg/g (ppt) of serum lipid. The CALUX-TEQ levels measured in this study indicate that there is low activity relative to the concentrations of PCBs, PCDFs, and dioxins measured in maternal serum and cord blood in previous studies1,8 by using traditional analytical methods. For example, maternal serum PCBs and PCDFs levels in excess of 25 ppb were documented in the Taiwanese cohort,1 whereas PCB and dioxin levels in maternal plasma and cord blood from The Netherlands were 2.2 and 0.45 ppb, respectively.8 Slightly higher concentrations of PCBs have been documented in fish eaters in the Michigan cohort compared with nonfish eaters, 6.1 ppb versus 4.1 ppb, respectively.3 The lower levels of dioxin-like activity documented in the current study may be the result of different analytical methods. Whereas prior studies have carried out congener-specific analyses and summed PCBs and dioxins to arrive at a TEQ value,1,3,8 we and others have quantified dioxin-like activity using a gene reporter assay.36 The DR-CALUX assay provides a composite measure of the activity of all chemicals present in the serum that act through the AhR and thus provides different but equally important information for population health risk assessment. Despite a similar analytical approach, our results are also lower than the CALUX-TEQ measured in women residing in Seveso, Italy, where plasma concentrations were 25.4 ppt lipid adjusted and a range of 0 to 127.6 ppt.36 Because of the accidental release of dioxins into the environment, serum dioxin-like activity in the Seveso, Italy, cohort would be expected to be higher than in our study population that is representative of a population with background exposure only. Previous studies have demonstrated that PCBs and dioxins accumulate with age and levels decline with parity and duration of breast feeding.42 In the current study, pregnant women were recruited from the population of pregnant women undergoing a second-trimester amniocentesis for advanced maternal age or maternal anxiety. The women making up our study population were close in age and covered only a 10-year span ranging from 34 to 44 years. Although our study population is not broadly representative of the general population of all pregnant women, the subset of pregnant women participating in this study represent the age-range of a substantial portion of pregnant women as more women postpone childbearing with a consequent increase in the proportion of births (29% in 1986 and 45% in 1999) now occurring among women older than 30 years.43 Similarly the proportion of first births in women under the age of 24 years has steadily declined from 46% to 24% between 1971 and 1999, whereas the proportion

Foster, Holloway, and Hughes of first births in women above the age of 25 has increased for all age groups.43 Furthermore, compared with younger gravid women, our study population would be expected to be at increased risk for higher body burdens of persistent environmental toxicants and thus potential adverse health outcomes. Therefore, if anything, our study design would be expected to bias our results in favor of finding an association between exposure and effects in this study population. However, there was no effect of age, maternal BMI, parity, or duration of breast feeding on the dioxin-like activity measured in these second-trimester maternal serum samples. We interpret these results to indicate that background levels of dioxin-like chemicals are low in women in this region of Canada. Furthermore, we speculate that lifestyle, age, and family planning and breast feeding practices in our study population are too similar to permit for differences in exposure to these environmental toxicants to be detectable. PCBs, PCDFs, and PCDDs are lipophylic compounds, and thus exposure to these environmental toxicants are primarily through the diet44 with levels measured in fast food being higher than in fresh food.45 Similarly, dioxin-like compounds such as benzo[a]pyrene46 are also present in cigarette smoke and thus maternal diet and smoking habits were assessed in this study. In this study no relationship between serum dioxin-like activity and diet could be demonstrated. Although all but 4 study subjects reported consuming fast food meals, the number of fast food meals consumed on a monthly basis was less than 1 per week. Moreover, in this study, few study subjects reported consuming Great Lakes fish and the number of Great Lakes fish meals consumed per month was also low. Thus, although the diet is the principal route of exposure to PCBs and dioxins, the subjects in this study did not consume appreciable amounts of foods thought to be high in these contaminants. In addition, the percentage of women reporting to be daily cigarette smokers is consistent with a recent survey.47 However, the amount smoked may be too low to significantly increase the concentration of dioxin-like chemicals and thus their activity in maternal serum. We further speculate that trace levels of dioxin-like chemicals are so widespread in the environment and the diet that background levels in the population will be very similar. Although conflicting reports have appeared in the literature,48,49 PCB and dioxin exposure has been associated with increased serum levels of TSH and decreased T4 in several animal studies50-52 and thus may provide a mechanism for dioxin-like chemical-induced neurotoxicity. However, in the current study we were unable to find any relationship between maternal serum dioxinlike activity and circulating levels of either TSH. Our results are in contrast with those from children in the Dutch cohort53 but are in agreement with umbilical cord blood samples from the North Carolina cohort.54

1905 Divergent results can be explained by the greater exposures documented in the Dutch cohort compared with those of the current study. Discrepant results may also be a function of using different sampling paradigms such as umbilical cord blood54 versus second-trimester maternal serum as in our cohort. However, our results are in agreement with an earlier study8 in which infant plasma in the second week after birth was analyzed. Our report is important because pregnant women may present for prenatal care with previously undiagnosed clinical hypothyroidism55 or with subclinical hypothyroidism, in which maternal thyroptropin levels are above a statistically defined reference range while maternal thyroxine levels are within the reference range.56 Subclinical maternal hypothyroidism has been reported to occur in 2.3% of pregnancies.57 Although appropriate clinical management of subclinical hypothyroidism in pregnancy remains controversial,57 accurate determination of maternal thyroid function will clearly be increasingly important for both researchers and clinicians. In summary, in this study we found that the vast majority of women studied had measurable levels of dioxin-like activity in their second-trimester serum. Background levels of dioxin-like activity measured in the current study could not be associated with maternal age, BMI, parity, duration of breast feeding associated with previous births, and cigarette smoking. Moreover, maternal diet did not provide insight into the sources of the background levels of dioxin-like activity documented in this study. Hence, we conclude that background levels of dioxin-like activity are low in our Canadian population and are not associated with adverse effects on maternal thyroid function.

Acknowledgments We gratefully acknowledge to time, patience, and effort of the study subjects for agreeing to participate in this study and the recruitment expertise of our study nurse Ms Mary Louis Beecroft. We also acknowledge the assistance of undergraduate students Ms Catherine MacPherson and Mr Zain Kassam for data entry and statistical analyses.

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