3. CONTENTS
3.1. Introduction
Over the last decades, the incidence of gestational diabetes (GD) is increasing and currently one
in seven births is affected by GD1. This is a significant public health concern, as GD is associatedwith dramatic adverse consequences, including preeclampsia; birth complications, cesareandelivery, as well as long-term risk of type 2 diabetes, obesity, and cardiovascular disease for boththe mother and child2. Thus, it is critical to identify alternative root causes of GD3. Given thecompelling evidences already available between exposure to phenolic compounds with T2D4 andthe extensive exposure of pregnant women to these chemicals, this study aims to evaluate thelevels of these chemicals in pregnant women with and without GD.

3.2. Procedure
3.2.1. SamplingThis study was approved by the Ethics Committee of Centro Hospitalar do Baixo Vouga (in which
Aveiro Hospital is included) and by the Portuguese National Data Protection Agency. Samples
from 45 pregnant women were collected. From these women, morning spot urine samples werecollected during the first consultation of the 1st trimester. An aliquot of each sample wastransported to CMES in cool conditions and preserved at -20oC until analysis.

3.2.2. Chemical analysis
At CMES, levels of triclosan, triclocarban, parabens (methyl, ethyl, propyl, butyl), benzophenones
2-OH-4-MeO-BP (BP-3); 2,4-diOH-BP (BP-1); 2,2'-diOH-4-MeO-BP (BP-8); 2,2',4,4'-tetraOH-BP(BP-2); 4-OH-BP) and bisphenols (BPA, BPB, BPC, BPF, BPS, BPZ, BPAF, TBBPA) were quantified inurine samples. The target compounds were analyzed following the protocol described by Kunisue
et al (2010)9 after some modifications and optimization by the team members (see LaMer
2017/18 report). Briefly, 50 µL of Internal Standards (ISs) Mixture was added to each sample
after being hydrolised with β-glucuronidase/aryl-sulfatase for 16h at 370C. Afterwards, cold
methanol, ultrapure water and ammonia solution (5% NH4OH) were added. The samples werethen loaded into a pre-conditioned OASIS MAX cartridge (MTBE, Methanol and ultrapure water).
Prior to elution with formic acid:MTBE:methanol = 0.2: 3: 7 (v/v/v), the cartridges were washedwith 5% NH4OH, 5% NH4OH in methanol, Milli-Q-water: methanol = 0.2: 6: 4 (v/v/v) andafterwards completely dried for 15 min. The eluted target compounds were evaporated to2

dryness under nitrogen flux and re-dissolved with acetonitile and 50 µL of mixture of naproxen13

C,d3 and ketoprofen-13C,d3 (20 ng/mL each) was added. The samples were preserved in amber

LC glass vials at 40C and before injection into the LC-MS/MS, Milli-Q water was added to a final
volume of 1mL.
The internal standard (ISs) mixture contains 100 ng/mL of Triclosan-13C6, 20 ng/mL of
Triclocarban-13C6, 20 ng/mL of Methyl paraben-13C6 and 20 ng/mL of Butyl paraben-13C6).
The activity of β-glucuronidase/aryl-sulfatase was 290 units per mL of urine. The solution was
prepared twice a week by adding 4.7 ml of 1.0 mol/L ammonium acetate; 5.3 mL of 1.0 mol/Lacetic acid and 50 µL of β-glucuronidase/aryl-sulfatase solution (116,000 units/mL). The crude
mixture of β-glucuronidase/sulfatase from Helix pomatia (Type HP-2, aqueous solution, 116,000
units/mL glucuronidase and 1020 units/mL sulfatase) was purchased from Sigma-Aldrich (St.
Louis, MO, USA); (G7017).

3.4. Results
Of the 19 compounds analyzed, five were not detected in any sample being always bellow theirrespective detection limit (Triclocarban, Bisphenols AF, B, C and Z). Overall, it was possible todetect at least two EDCs in each sample, with an average of 6.4 compounds per sample. The
most frequent compounds were Bisphenol-F and Methylparaben, detected in 97.8% of thesamples, followed by Benzophenone-1 and Ethylparaben detected in 93.3 and 91.1% of thesamples (Figure 1).

3% detection
1009080706050403020100

Figure 1. Percentage of detection of each EDC in all the urine samples analyzed (n=45).

Generally, the highest levels were associated with benzophenone-3 (12.9 ng/µg creatinine),
methylparaben (3.4 ng/µg creatinine) and triclosan (1.1 ng/µg creatinine). Of the parabens, andas expected, methylparaben was the one with the highest concentrations (Figure 2).

Figure 2. Boxplot summarizing the values of parabens and antimicrobilas (left panel) andbisphenols (right panel) detected in the analyzed urine samples.

As for the antimicrobials only triclosan was detected with values ranging from <MDL (3.91x10-4ng/µg creatinine) up to 1.1 ng/µg creatinine. In what concerns the benzophenon UV filters,
benzophenone-1 was detected in 93.3% of the samples with values ranging between 3.91x10-44ng/µg creatinine and 1.1 ng/µg creatinine and benzophenon-3 was detected in 73.3% of thesamples with one sample registering values as high as 12.85 ng/µg creatinine.
Of the bisphenols, interestingly, BPA was not the most prevalent compound nor the one that
registered the highest concentrations. In fact, it was detected in 40% of the samples with anaverage value of 0.0071 ng/µg creatinine whereas its substitute BPF was detected in 97.8% of
the samples with an average value of 0.0186 ng/µg creatinine.

3.5. Future perspectives
Clinical data from all the patients is now being computed and analyzed. The obtained resultsfrom the chemical analysis will be analyzed by means of multivariate statistics and correlatedwith clinical data. Sampling continues in order to obtain a more robust data set.

3.6. Achievements
Oral Communications by Invitation
Sousa ACA (2019) Environmental contaminants and endocrine disruptors: a global
perspective on main sources, exposure pathways and health effects. Ricardo Jorge
Seminar, National Institute of Health Dr. Ricardo Jorge, 24 October 2019, Lisbon, Portugal.

Sousa ACA (2019) The interference of environmental contaminants on the thyroid gland.
VII Jornadas de Endocrinologia, Diabetes e Nutrição de Aveiro, 23-24 May, Aveiro,Portugal.

Oral Communications
Souto-Miranda S, Melo-Dias S, Valente C, Freite C, Sousa AM, Sousa ACA, Marques A(submitted) Patients’ perspectives on exposure to environmental contaminants: amissing piece in pulmonary rehabilitation? 30th European Respiratory Society AnnualCongress 2020, 5-9 September 2020, Vienna, Austria

e Silva FA, Rocha B, Santo MJ, Neves MC, Sousa ACA, Freire MG (2019) New opportunities
brought about by supported ionic liquids within the removal of anticancer drugs fromaqueous solutions. BPP 2019, Biopartitioning & Purification Conference, 11-13November 2019, Guarujá, SP, Brazil, p.29

Sousa ACA, Souto-Miranda S, Marques A, Valente C, Barros R, Taborda-Barata L, TanoueR, Kunisue T, Tanabe S, Pastorinho MR (2019) Endocrine Disrupting Chemicals in Patientswith Chronic Obstructive Pulmonary Diseases. PTIM 2019, 3rd International Caparica
5Conference on Pollutant Toxic Ions and Molecules, 4-7 November 2019, Caparica,Portugal, p. 97, ISBN 978-989-54470-3-9
Posters in Conferences/Workshops
Sousa ACA, Assunção R, Coelho SD, Kunisue T, Tanabe S, Pastorinho MR (2020) Dietaryexposure to Cadmium and the associated burden – results from a duplicate diet study inPortugal (2012). Glob 1st WG meeting & 2nd MC meeting Burden of Disease in Europe
Taking stock and moving forward. National Food Institute, Technical University ofDenmark, 18-19 February 2020, Copenhagen, Denmark, p. 21

Sousa S, Barros R, Pastorinho MR, Sousa ACA (2019) Association between obesity andmercury: preliminary results from the elegance study. XIV Annual CICS-UBI Symposium
2019, 4-5 July 2019, Covilhã, Portugal, p. 71

Sousa ACA, Souto-Miranda S, Marques A, Silva T, Henriques I, Barros R, Taborda-BarataL, Valente C, Tanoue R, Kunisue T, Tanabe S, Pastorinho MR (2019) Personal care products
in matched human and Environmental samples collected under the framework ofRESPIRA project. Jornadas CICECO, University of Aveiro, 11-12 June 2019, Aveiro,Portugal, p. 159

Peer-reviewed abstracts in international conferences
Sousa ACA, Assunção R, Coelho SD, Kunisue T, Tanabe S, Pastorinho MR (2020) Dietary
exposure to Cadmium and the associated burden – results from a duplicate diet study
in Portugal (2012). Glob 1st WG meeting & 2nd MC meeting Burden of Disease inEurope Taking stock and moving forward. National Food Institute, Technical Universityof Denmark, 18-19 February 2020, Copenhagen, Denmark, p. 21, https://www.burdeneu.net/docs/m1wg-abstract-book.pdf

Book chapters
Pastorinho MR, Sousa ACA (2020) Pets as sentinels of human exposure to neurotoxic
metals. In Pastorinho MR, Sousa ACA (Eds) Pets as Sentinels, Forecasters and Promotersof Human Health. Springer International Publishing AG, Switzerland, ISBN: 978-3-03030733-2, p.83-106; https://doi.org/10.1007/978-3-030-30734-9_5

Publications for science and technology dissemination
Sousa ACA (2019) “The interference of environmental contaminants on the thyroidgland.” Jornal das VII Jornadas de Endocrinologia, Diabetes e Nutrição de Aveiro, p. 9.

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References:(1) International Diabetes Federation; Federation, I. D., Ed.; International Diabetes Federation:
2018.
(2) Kessous, R.; Shoham-Vardi, I.; Pariente, G.; Sherf, M.; Sheiner, E. Heart 2013, 99, 1118.
(3) Ehrlich, S.; Lambers, D.; Baccarelli, A.; Khoury, J.; Macaluso, M.; Ho, S.-M. Amer J Perinatol
2016, 33, 1313.
(4) (4) Song, Y.; Chou, E. L.; Baecker, A.; You, N.-C. Y.; Song, Y.; Sun, Q.; Liu, S. Journal of Diabetes
2016, 8, 516.

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