Causes of Placental Transfer of Mercury in Women: Proposal

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16 Apr 2018

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Placental transfer of mercury in women from Aveiro district: influence of lifestyle and environmental factors

Keywords: Biomarkers; Epidemiology; Genotoxicity; Maternal transfer; Mercury

 

CONTEXT (1000 words):

Estarreja is one of the cities surrounding the Ria de Aveiro lagoon system in the Aveiro district. This aquatic system integrates a very important ecosystem that provides goods and services to the human populations, including supporting, provisioning, regulating and also cultural services. All these are closely connected to human well-being and sustainability, and depend on the ecosystem structure, mainly on the abiotic and biotic constituents, which are closely related to the natural processes occurring and the external inputs into the system.

Natural and human origin drivers will be key points for the maintenance of ecosystems, and for the goods and services that they can provide to human populations. Industry is a key motor for Estarreja region, although representing also a threat to the environment and human populations. It is widely known that mercury (Hg) contamination is an important issue in the Ria de Aveiro region, especially in areas nearby the Largo do Laranjo area, where mercury in sediments can reach 50 g Hg/g, being this partly attributed to effluents from chlor-alkali plants (REF, Abreu ou pereira et al 1997). Although the highest concentrations are found in this area, natural occurring processes like stronger tidal currents and bottom resuspension can spread this contamination, although at lower levels, throughout the channels of the lagoon. This can lead to fish contamination and related biomagnification through the food web, ending up on human exposure via ingestion. Most of mercury in diet is usually attributed to the presence of methylmercury in fish and seafood. Another problem in this region regarding Hg, although at a lower scale, can be considered air emissions which can harm ecosystems by deposition but most importantly constitute another route of uptake for humans. This will lead to a primary toxicity regarding exposure by lungs, skin and eyes, inducing symptoms related to the respiratory and digestive systems, but also related to the central nervous system. Another problem closely related to primary toxicity was related to dental practices. Some decades ago, this primary toxicity was very common on people with dental amalgam, as they were constituted mainly by several metals (e.g. Ag, Cu) including Hg. With time, metals were released by dissolution and entered in the human systems causing toxicity. Secondary toxicity can further include problems related to the nervous systems in conjunction with the cardio-vascular system. Upon entrance in the body, Hg can be transported and accumulated depending on the exposure route but also on the elemental form present. It is known that elemental Hg (Hg0) and organic Hg (e.g. methyl-mercury) can cross the blood-brain barrier and can be found in the placenta and breast milk, contrary to inorganic Hg which can mainly affect kidneys and the gastrointestinal tract (chapter 2- env-health.org).

To monitor Hg accumulation in humans, several studies have been carried out worldwide, using as assessment matrices blood, urine, fingernails, and hair, and in cases where studies are devoted to mother-baby transfer samples of breast milk, fetal blood, umbilical cord blood and placenta are often assessed.

Accumulation studies are crucial to assess human risk because they closely relate the bioaccessible fraction of chemicals in the environment (exposure) with effects. In addition early warning tools like the measurement of enzymatic biomarkers related to oxidative stress, neurotoxicity or DNA damage can improve and complement this monitoring programmes, providing information on how detoxification is been carried out (if the case), toxicity at the central nervous system or DNA damage are prone to occur. When effects are not visible, they can be further predicted by this kind of approaches and potentially prevents worst case scenarios of exposure and effects.

PURPOSES (1000 words)

This project aims at assessing the accumulation of Hg by woman living in the Aveiro district, and the potential mother-baby transfer during pregnancy. In addition to Hg measurements, related oxidative stress, neurotoxicity and DNA damage effects will also be evaluated in both mother and baby tissues and blood.

Therefore several questions and hypothesis will be raised in the several phases of the project. The first question regards the accumulation of Hg by pregnant woman, and relates to daily habits. For that, a questionnaire is available to be filled in by the parturient (with the help of a medical doctor), where habits are questioned and recorded. Food type and frequency, smoking habits, and cosmetic application are some of the items included and that will be crossed with Hg measurements on hair and blood samples. The second question is more devoted to the Hg transport in the body, and therefore regards on how effective is the mother-baby transfer, and to what extent this can occur. For that, placenta measurements, in three distinct areas (membrane, contact area with the baby and contact are with the mother), will be carried out in addition to umbilical blood and tissues from the umbilical cord. This approach will also provide the opportunity to correlate Hg concentrations on the several samples collected to minimize, when possible, time and money consuming for collecting samples in monitorization assets. The third question relates more closely exposure to effects. Therefore, by evaluating oxidative stress, neurotoxicity and DNA damage we will infer on how the Hg accumulation is being unraveled by physiological processes in the organism, and if there are already some effects.

By integrating the three questions and their respective answers, this project will provide an overview of the accumulation of Hg on pregnant woman from Estarreja and from the Aveiro district, relating that to a potential accumulation in the baby and future induced effects.

METHODOLOGY (1000 words)

The methodology of this project presented below was approved on September 2014 by the Ethical Committee of the Hospital Infante Dom Pedro, Aveiro (HIDP), Portugal, (document attached) and will be performed in collaboration with the Obstetrics and Gynecology service from this hospital.

  1. Study population

Fifty women in labor will be selected for the present study when admitted for delivery to the Obstetrics and Gynecology service of HIDP. All selected participants will be asked to give informed consent (document in attachment) before filling of the questionnaire and collection of samples.

  1. Questionnaire

Ver Valent et al. (2013)

  1. Sampling and preservation of biological samples

The collection of samples will be performed by the Obstetrics and Gynecology nurse team of HIDP and kept at 4ºC. Samples will then be transported to the Department of Biology, University of Aveiro (distance of 500 m) on a daily basis in refrigerated conditions, for further processing and preservation for Hg quantification and/or analyses of neurotoxic, oxidative stress and genotoxic biomarkers.

  1. Hair

Hair samples with at least 2.5 cm long will be collected from each parturient in the occipital region and near the root with a sterile stainless steel scissor. Samples will be kept in 2 mL microtubes until further processing. In the laboratory, the hair samples will be cut into 2 cm segments and subjected to washing according to standard procedures recommended by the International Atomic Energy Agency (REFª?): (1) washing with acetone, (2) three times washing in ultra-pure water and (3) a final wash in acetone. Then the samples will be dried overnight at 40°C for Hg quantification.

  1. Parturient and cord blood

Blood samples of about 2.7 ml will be collected in EDTA containing tubes, both from each parturient and their respective umbilical cord. Aliquots of total blood will be maintained at -20ºC until total Hg quantification. The remaining volume of blood will be divided in aliquots and centrifuged (10,000 g; 5 minutes). Plasma and blood cells will then be maintained at -80ºC until enzymatic biomarker analysis.

  1. Placenta

Just after delivery, a section of placenta will be collected and maintained in 0.9% NaCl at 4ºC. The placenta will then be dissected and divided in different sections for total Hg quantification, namely, the membrane, maternal and fetal side portions, that will be weighted and preserved at -20ºC until analysis. Part of the placenta (excluding membranes) will be maintained at -80ºC for enzymatic biomarkers analysis and comet assay analysis.

  1. Quantification of Mercury

For the quantification of mercury in the different biological matrices, the samples will be lyophilized, homogenized, weighted and then analyzed by atomic absorption with thermal decomposition of the sample (AMA-254 LECO). Quality control will be assured by analysis, in parallel, of certified reference samples (Bovine Muscle, ERM® –BB184).

  1. Biomarkers of neurotoxicity and oxidative stress

The enzymatic biomarkers below described will be analyzed in the aliquots of cord blood, mother blood and/or samples of placenta previously collected.

Acetylcholinesterase (ChE) activity is a biomarker of neurotoxicity and will be determined in blood cells and in placenta samples. Briefly, samples will be homogenized in phosphate buffer 0.1 M (pH 7.2) and centrifuged at 6000 rpm during 5 minutes. The enzymatic reaction will be performed in the obtained supernatants as described by Ellman et al. (1961) adapted to microplate reader by Guilhermino et al. (1996).

Lactate dehydrogenase (LDH) activity will be determined in blood plasma and placenta. Plasma will be diluted in Tris / NaCl (0.1 M, pH 7.2) and LDH activity will be determined according to the method described by Vassault (Vassault 1983) and adapted to microplate by Diamantino et al. (2001).

Lipid peroxidation, as a measure of oxidative stress damage, and the activity of enzymes involved in oxidative stress response will be quantified in blood plasma and placenta. Lipid peroxidation will be determined through the quantification of thiobarbituric acid reactive substances (TBARS) according to the methodology described by Bird and Draper (1984) and Ohkawa et al. (1979). Glutathione S--transferases (GST) activity will be determined according to the method described by Habig et al. (1974). Quantification of glutathione peroxidase (GPx) enzymatic activity will be performed through the method of Mohandas et al. (1984). Finally, catalase (CAT) activity will be quantified following the decomposition of H2O2 at 240 nm according to the method of Claiborne (1985).

  1. Genotoxicity assessment

The assessment of DNA damage in placental tissue will be performed through the Comet assay. Suspension of cells from placental tissue will be obtained and collected in ice-cold PBS with 20 mM EDTA and then centrifuged at 4ºC. The pellet will be suspended in FBS (fetal bovine serum) with 10% DMSO and frozen until further analysis. A fluorescence microscope (Olympus BX41TF, China, equipped with an excitation filter BP 546/12 nm and a barrier filter of 590 nm) will be used for comet assay. DNA damage will be identified as described by Duthie e Collins (1997), using a scale of 0 (no damage) to 4 (severe damage) to classify at least 100 cells per sample.

References

Bird, R.P., Draper, H.H., 1984. Comparative studies on different methods of malonaldehyde determination. Methods in Enzymology 105, 299-305.

Clairborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), CRC Handbook of Methods in Oxygen Radical Research. CRC Press, Boca Raton, FL.

Cortes Toro E, De Goeij J, Bacso J, Cheng Y-D, Kinova L, Matsubara J, Niese S, Sato T, Wesenberg G, Muramatsu Y, Parr R (1993) The significance of hair mineral analysis as a means for assessing internal body burdens of environmental pollutants: Results from an IAEA Co-ordinated Research Programme. J Radioanal Nucl Chem 167(2): 413–421

Diamantino, T.C., Almeida, E., Soares, A., Guilhermino, L., 2001. Lactate dehydrogenase activity as an effect criterion in toxicity tests with Daphnia magna straus. Chemosphere 45, 553-560.

Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88-90.

Ferreira, N. G. C., Santos, M. J. G., Domingues, I., Calhôa, C. F., Monteiro, M., Amorim, M. J. B., Loureiro, S. (2010). Basal levels of enzymatic biomarkers and energy reserves in Porcellionides pruinosus. Soil Biology and Biochemistry, 42(12), 2128–2136.

Guilhermino, L., Lopes, M.C., Carvalho, A.P., Soares, A., 1996. Acetylcholinesterase activity in juveniles of Daphnia magna Straus. Bulletin of Environmental Contamination and Toxicology 57, 979-985.

Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases as first enzymatic step on mercapturic acid formation. Journal of Biological Chemistry 249, 7130-7139.

Mohandas, J., Marshall, J.J., Duggin, G.G., Horvath, J.S., Tiller, D.J., 1984. Low activities of glutathione e related enzymes as factors in the genesis of urinary-bladder cancer. Cancer Research 44, 5086-5091.

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351-358

Vassault, A., 1983. Lactate dehydrogenase. In: Methods of Enzymatic Analysis. Academic Press, New York.

Vieira, H. C., Morgado, F., Soares, a M. V. M., & Abreu, S. N. (2013). Mercury in scalp hair near the Mid-Atlantic Ridge (MAR) in relation to high fish consumption. Biological Trace Element Research, 156(1-3), 29–35.

Bird R. & Draper H. (1984). Comparative studies on different methods of malonaldehyde determination. Methods in enzymology, 105, 299.

Clairborne A. (1985). Catalase activity. CRC handbook of methods for oxygen radical research, 1, 283-284.

Diamantino T.C., Almeida E., Soares A.M. & Guilhermino L. (2001). Lactate dehydrogenase activity as an effect criterion in toxicity tests with< i> Daphnia magna</i> straus. Chemosphere, 45, 553-560.

Ellman G.L., Courtney K.D. & Featherstone R.M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical pharmacology, 7, 88-95.

Guilhermino L., Lopes M., Carvalho A. & Soares A. (1996). Acetylcholinesterase activity in juveniles of Daphnia magna Straus. Bulletin of environmental contamination and toxicology, 57, 979-985.

Habig W.H., Pabst M.J. & Jakoby W.B. (1974). Glutathione S-transferases the first enzymatic step in mercapturic acid formation. Journal of biological Chemistry, 249, 7130-7139.

Mohandas J., Marshall J.J., Duggin G.G., Horvath J.S. & Tiller D.J. (1984). Low activities of glutathione-related enzymes as factors in the genesis of urinary bladder cancer. Cancer Research, 44, 5086-5091.

Ohkawa H., Ohishi N. & Yagi K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry, 95, 351-358.

Vassault A. (1983). Lactate dehydrogenase. UV-method with pyruvate and NADH. Methods of enzymatic analysis, 3, 118-126.



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