Associations of Fetal Growth Outcomes with Measures of the Combined Xenoestrogenic Activity of Maternal Serum Perfluorinated Alkyl Acids in Danish Pregnant Women.

BACKGROUND: Higher concentrations of single perfluorinated alkyl acids (PFAAs) have been associated with lower birth weight (BW), but few studies have examined the combined effects of PFAA mixtures. PFAAs have been reported to induce estrogen receptor (ER) transactivity, and estrogens may influence human fetal growth. We hypothesize that mixtures of PFAAs may affect human fetal growth by disrupting the ER. OBJECTIVES: We aimed to study the associations between the combined xenoestrogenic activity of PFAAs in pregnant women's serum and offspring BW, length, and head circumference. METHODS: We extracted the actual mixture of PFAAs from the serum of 702 Danish pregnant women (gestational wk 11­13) enrolled in the Aarhus Birth Cohort (ABC) using solid phase extraction, high-performance liquid chromatography (HPLC), and weak anion exchange. PFAA-induced xenoestrogenic receptor transactivation (XER) was determined using the stable transfected MVLN cell line. Associations between XER and measures of fetal growth were estimated using multivariable linear regression with primary adjustment for maternal age, body mass index (BMI), educational level, smoking, and alcohol intake, and sensitivity analyses with additional adjustment for gestational age (GA) (linear and quadratic). RESULTS: On average, an interquartile range (IQR) increase in XER was associated with a [Formula: see text] [95% confidence interval (CI): [Formula: see text], [Formula: see text]] decrease in BW and a [Formula: see text] (95% CI: 0.1, 0.5) decrease in birth length. Upon additional adjustment for GA, the estimated mean differences were [Formula: see text] (95% CI: [Formula: see text], 4) and [Formula: see text] (95% CI: [Formula: see text], 0.0), respectively. CONCLUSION: Higher-serum PFAA-induced xenoestrogenic activities were associated with lower BW and length in offspring, suggesting that PFAA mixtures may affect fetal growth by disrupting ER function. https://doi.org/10.1289/EHP1884.

Receptor activities of persistent pollutant serum mixtures and breast cancer risk.

Studies on associations between persistent organic pollutants (POPs) and breast cancer risk are inconclusive. The majority of studies have evaluated the effect of single compounds, without considering multiple exposures to and interactions between different POPs. The present study aimed at evaluating breast cancer risk related to combined effects of serum POP mixtures on cellular receptor functions. Data on breast cancer cases (n = 77) and controls (n = 84) were collected among Greenlandic Inuit women. Serum mixtures of lipophilic POPs (lipPOPs), perfluoroalkyl acids (PFAAs) and dioxin-like POPs were extracted. The effect of the mixture extracts on the estrogen receptor (ER), androgen receptor (AR) and aryl hydrocarbon receptor (AhR) was determined using cell culture reporter gene assays. The serum mixtures were analyzed alone and upon co-exposure with natural receptor ligands to determine agonistic and antagonistic/competitive activity. We found that the frequency of lipPOP mixtures eliciting no, decreasing, or agonizing xenoandrogenic effect differed by breast cancer status. Using lipPOP mixtures with no effect on AR as reference, the mixtures with decreasing effects reduced breast cancer risk (OR: 0.30 (0.12; 0.76)). The AhR-toxic equivalent of serum mixtures was significantly lower in cases than in controls, and a reduced breast cancer risk was found when comparing the third tertile to the first (OR: 0.34 (0.14; 0.83)). We found no association between the xenoestrogenic activities of lipPOPs or PFAAs and breast cancer risk. Serum lipPOP mixtures are hormone disruptive and may influence breast cancer risk, whereas PFAAs seem to influence breast cancer risk through other pathways.

Temporal trends of lipophilic persistent organic pollutants in serum from Danish nulliparous pregnant women 2011-2013.

The use of the lipophilic persistent organic pollutants (POPs) including polychlorinated biphenyls (PCBs) and several organochlorine pesticides (OCPs) has been prohibited for more than 30 years. In this study, we present the temporal trends of the lipophilic POP serum concentrations in Danish nulliparous pregnant women between 2011 and 2013. We randomly selected 197 pregnant women (gestational age 11-13) from the Aarhus Birth Cohort. The concentrations of the lipophilic POPs in the serum samples were analyzed using gas chromatography. The concentrations were corrected for total serum lipids. The statistical analysis was performed by regression analysis with adjustment for age, BMI, gestational age at blood draw, and smoking status. The serum concentrations of PCB 118, 138, 153, 156, 170, 180, 187, and hexachlorobenzen, trans-nonachlor, ß-hexachlorocyclohexane (ß-HCH), and p,p'-dichlorodiphenyldichloroethylene were lower in 2013 than in 2011. However, the oxychlordane concentration was lowest in 2011. The serum levels of most lipophilic POPs followed downward trends during the study period, which was expected, as these compounds has been banned for many years. The upward trend of oxychlordane was unexpected and presumably a chance finding.

Maternal serum concentrations of perfluoroalkyl acids in five international birth cohorts.

BACKGROUND: Perfluoroalkyl acids (PFAAs) are persistent and bioaccumulating compounds, which are spread all over the globe. We aimed to compare the PFAA concentrations in serum from pregnant women in five birth cohorts from four countries (Denmark, China, Norway, and Greenland). METHODS: Serum samples were obtained from the following five birth cohorts including a total of 4718 pregnant women: the Danish National Birth Cohort (DNBC, years 1996-2002, Denmark), the Aarhus Birth Cohort (ABC, years 2008-2013, Denmark), the Shanghai Birth Cohort (SBC, years 2013-2015, China), the Northern Norway Mother-Child Contaminant Cohort (MISA, years 2007-2009, Norway), and the Greenlandic Birth Cohort (ACCEPT, years 2010-2013, Greenland). The samples were analyzed using liquid chromatography triple-quadrupole mass spectrometry. To ensure comparability, all samples except for the MISA samples were measured in the same laboratory. We adjusted the log-transformed PFAA concentrations for age and parity using analysis of covariance. RESULTS AND DISCUSSION: The geometric mean (GM) of the summed concentrations of the seven most abundant PFAAs (∑PFAA) was 35ng/mL in the DNBC, 25 ng/mL in the SBC, 18ng/mL in the ACCEPT, 12ng/mL in the MISA cohort, and 12ng/mL in the ABC. The DNBC concentration was highest presumably because these samples were taken in earlier years (i.e. 1996-2002) than the samples from the other cohorts (i.e. 2007-2015), and at a time when the production of PFAAs were at the highest. When excluding the DNBC samples, we found that the concentrations of all the perfluorinated sulfonic acids (PFSAs) and one of the four perfluorinated carboxylic acids (PFCAs) were highest in the Greenlandic women, whereas the other three PFCAs were highest in the Chinese women. CONCLUSION: The concentration and composition of serum PFAAs were similar for the Danish ABC women and the Norwegian MISA women but were otherwise different across the cohorts. The different exposure profiles might partly be related to differences in lifestyle and diet. As the concentrations and compositional patterns vary between the countries, we suggest that the health implications associated with high PFAA exposure might also differ between the countries.

Activation of the estrogen receptor by human serum extracts containing mixtures of perfluorinated alkyl acids from pregnant women.

Humans are exposed to a wide variety of perfluorinated alkyl acids (PFAAs). Several studies have found xenoestrogenic activity of single PFAAs. Studies on mixture effects of the PFAAs are however sparse. In the present study, we aimed to determine the xenoestrogenic activity in human serum extracts containing mixtures of PFAAs. Recently we developed a method to extract the PFAAs from human serum with simultaneous removal of endogenous hormones and interfering steroid metabolites. We used this method to extract the PFAAs from serum of 397 Danish nulliparous pregnant women followed by analysis of estrogen receptor (ER) transactivation using MVLN cells carrying an estrogen response element luciferase reporter vector. Using 17ß-estradiol (E2) concentration-transactivation curves, we calculated the estradiol equivalents (EEQ) for the extracts containing the PFAAs. Fifty-two percent of the PFAA serum extracts agonized the ER transactivation, and 46% enhanced the E2-induced ER transactivation. We found positive linear concentration-response associations between the ER transactivation and the PFAA serum levels. For the relatively few PFAA extracts that antagonized the ER in the presence of 24 pM E2 (n=38, 10%), we found inverse linear associations between the ER transactivation and the PFAA serum levels. The results indicated that the serum extracts induced the ER in a non-monotonic concentration dependent manner. The median EEQ of the extracts containing the PFAAs corresponds to the effect of 0.5pg E2 per mL serum. In conclusion, we observed that most of the extracts containing the PFAA mixtures from pregnant women's serum agonized the ER and enhanced the E2-induced effects in non-monotonic concentration-dependent manners.

Determinants of serum levels of perfluorinated alkyl acids in Danish pregnant women.

Humans are exposed to perfluorinated alkyl acids (PFAAs) from food, drinking water, air, dust, and consumer products. PFAAs are persistent and bio-accumulative. In the present study, we aimed to establish how the serum levels of PFAAs differ according to age, pre-pregnancy body mass index (BMI), previous miscarriages, educational level, country of birth, smoking, and alcohol intake. We included 1438 Danish pregnant nulliparous women from the Aarhus Birth Cohort. The women gave a blood serum sample between week 11 and 13 of pregnancy. Sixteen PFAAs were extracted from serum using solid phase extraction and analyzed by liquid chromatography/tandem mass spectrometry. Multivariable linear regression analysis was used to determine the associations between individual characteristics of the women and their levels of seven PFAAs that were detected in at least 50% of the samples. The total concentration of the PFAAs (∑PFAA) was higher in older women. On average, normal weight women had a higher ∑PFAA level than underweight, overweight, and obese women. Higher levels were also observed for women without previous miscarriages, women with a high educational level, women born in Denmark (as opposed to women born elsewhere but currently living in Denmark), non-smokers, and women who consumed alcohol before or during pregnancy. These associations were similar for all the studied PFAAs, although the levels of perfluoroundecanoic acid varied more across the categories of age, BMI, education, smoking, and alcohol consumption than any other PFAAs measured.

Time trends of perfluorinated alkyl acids in serum from Danish pregnant women 2008-2013.

We aimed to estimate the levels and time trends of perfluorinated alkyl acids (PFAAs) in serum of 1533 Danish pregnant nulliparous women between 2008 and 2013. The selection criterion of only including nulliparous women was chosen to avoid confounding from parity. The serum samples were analyzed for sixteen PFAAs using solid phase extraction and liquid chromatography tandem mass spectrometry (LC-MS/MS). We investigated the time trends for seven PFAAs, which were detected in more than 50% of the samples: perfluorohexane sulfonate (PFHxS), perfluoroheptane sulfonate (PFHpS), perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnA). We found that the serum levels of all seven PFAAs decreased during the period from 2008 to 2013; on average PFHxS decreased with 7.0% per year, PFHpS with 14.8%, PFOS with 9.3%, PFOA with 9.1%, PFNA with 6.2%, PFDA with 6.3%, and PFUnA with 7.1% per year. Adjustment for maternal age, body mass index (BMI), educational level and gestational age at blood sampling did not change the time trends much. To our knowledge, we are the first to report decreasing trends of PFNA, PFDA and PFUnA since year 2000, thereby indicating that the phase-out of these compounds are beginning to show an effect on human serum levels.

Estrone sulfate and dehydroepiandrosterone sulfate: Transactivation of the estrogen and androgen receptor.

Dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E1S) are two of the most abundant steroids in the human circulation. The enzyme steroid sulfatase (STS) cleaves the sulfate group of DHEAS and E1S leading to biosynthesis of endogenous hormones such as testosterone and estrone. In the current study we aimed at determining the effect of E1S and DHEAS on estrogen receptor (ER) and androgen receptor (AR) transactivation. Using luciferase reporter gene assays, the ER and AR transactivities of E1S and DHEAS were determined by direct cell exposure; as well as upon extraction from human serum using a method to extract perfluorinated alkyl acids (PFAAs). By direct cell exposure, both E1S and DHEAS transactivated the ER and the AR in dose-dependent manners. The DHEAS-induced AR transactivity could be abolished by the STS inhibitor STX64. Immunoassay analysis confirmed the presence of E1S and DHEAS in the serum PFAA extracts with mean recoveries below 2.5%. For the PFAA extracts of human male and female serum, only the AR was significantly transactivated. The AR transactivity of the sulfated steroids in the extracts was abolished by STX64 to obtain the net PFAA induced xenohormone transactivity, but further cleanup might be needed at high concentrations of E1S.

Extraction of perfluorinated alkyl acids from human serum for determination of the combined xenoestrogenic transactivity: a method development.

Humans are exposed to perfluorinated alkyl acids (PFAAs) through food, drinking water, consumer products, dust, etc. The human metabolism and excretion of the long-chain PFAAs is slow with half-lives up to 8.8years. Studies suggest that the PFAAs are potential endocrine-disrupting compounds that might affect human health. We developed a method for extraction of PFAAs from human serum with simultaneous removal of endogenous sex hormones. The developed method includes solid phase extraction, liquid/liquid extraction, HPLC fractionation and weak anion exchange. The method was validated by extraction of seven persistent PFAAs spiked to human male serum obtaining mean recoveries between 49.6% and 78.6%. Using an estrogen receptor (ER) transactivation luciferase reporter gene assay, analysis of the extracted PFAA serum fraction from three pregnant women showed the ER-active endogenous hormones were removed. The developed method was further documented by extraction of the PFAAs from the serum of 18 Danish pregnant women. The PFAA fraction from three of the 18 samples significantly induced the ER-transactivity. Upon co-exposure with the natural ER-ligand 17ß-estradiol (E2), 17 of the 18 PFAA fractions caused a significant further increase of the E2 induced ER-transactivity. In conclusion, we developed a method to extract PFAAs from human serum, and the method documentation suggested that PFAAs at the levels found in human serum can transactivate the ER.

Biomonitoring and hormone-disrupting effect biomarkers of persistent organic pollutants in vitro and ex vivo.

Persistent organic pollutants (POPs) include lipophilic legacy POPs and the amphiphilic perfluorinated alkyl acids (PFAAs). They have long half-lives and bioaccumulate in the environment, animals and human beings. POPs possess toxic, carcinogenic and endocrine-disrupting potentials. Endocrine-disrupting chemicals (EDCs) are compounds that either mimic or block endogenous hormones and thus disrupt the normal hormone homeostasis. Biomonitoring assesses the internal doses of a person to provide information about chemical exposures. Effect biomarkers assess chemicals potential to affect cellular functions in vivo/ex vivo. Human beings are exposed to complex mixtures of chemicals, having individually very different biological potentials and effects. Therefore, the assessment of the combined, integrated biological effect of the actual chemical mixture in human blood is important. In vitro and ex vivo cell systems have been introduced for the assessment of the integrated level of xenobiotic cellular effects in human beings. Ex vivo studies have shown geographical differences in bioaccumulated POP serum levels, being reflected by the combined biomarker effects of the complex mixture extracted from human serum. Xenohormone receptor transactivities can be used as an ex vivo integrated biomarker of POP exposure and effects. Epidemiological and in vitro/ex vivo studies have supported the potential impact of the combined effect of serum POPs on the activity of hormone and/or dioxin receptors as a risk factor for human health. With focus on hormone disruption, this MiniReview will give an update on recent POP-related endocrine-disrupting effects in vitro/ex vivo/in vivo and some related genetic data.