Team:USP-EEL-Brazil/Background
The Problem
Estrogens
Estrogens are classified as Endocrine Disruptor Chemicals (EDCs). They are exogenous compounds capable of interfering negatively with the functioning of the endocrine system, causing adverse effects on the growth, development and reproduction of living beings. According to the World Health Organization (WHO), there are more than 800 chemicals that can be classified as endocrine disruptors, such as natural and synthetic hormones, pharmaceuticals, plasticizers and pesticides. It is possible to classify estrogens into three categories:
• Natural and Synthetic estrogens: steroid hormones that can be separated into natural or synthetic origin. Naturals are those produced and secreted naturally by the body, while synthetics are developed for medical use in hormone replacement therapies and as contraceptive methods. • Xenoestrogens: synthetic substances used in industrialized products that mimic the action of estrogens in the body. Among these substances are: polychlorinated biphenyls, flame retardants (mainly brominated diphenyl ethers), phthalates, surfactants, plasticizers and some pesticides. • Phytoestrogens: compounds such as sitosterol and isoflavonoids, naturally produced by plants, being found in high concentrations in soybean, alfalfa and other vegetables.Our Focus
Our project focused on seven estrogens: three naturals, one synthetic and three xenoestrogens. Here are some features of our target estrogens:
Natural and Synthetic estrogens:
Estrone (E1): is a steroid sex hormone found both in male and female. It is one of three major endogenous estrogens and it is synthesized from cholesterol and secreted mainly from the gonads, though they can also be formed from adrenal androgens in adipose. Estrone can be converted into estradiol and serves mainly as a precursor or metabolic intermediate of estradiol.
β-Estradiol (E2): is another steroid hormone and the major female sex hormone. It is involved in the regulation of the estrous and menstrual female reproductive cycles. Estradiol is responsible for the development of female secondary sexual characteristics and it also has important effects in many tissues. Though estradiol levels in men are much lower compared to those in women, estradiol has important roles in men as well.
Estriol (E3): is a weak estrogen and a minor female sex hormone. Levels of estriol in women who are not pregnant are almost undetectable. However, during pregnancy, estriol is synthesized in very high quantities by the placenta and is the most produced estrogen in the body by far.
17α-Ethinylestradiol (EE2): is an estrogen medication which is used widely in birth control pills in combination with progestins. It is also occasionally used as a component of menopausal hormone therapy for the treatment of menopausal symptoms. It is a synthetic derivative of estradiol and has greatly improved bioavailability when taken by mouth, is more resistant to metabolism, and shows relatively increased effects in certain parts of the body like the liver and uterus.
Xenoestrogens:
Bisphenol-A (BPA): is an organic chemical that forms the basic-intermediate unit of high-performance polymers and coatings, mainly polycarbonate plastics and epoxy resins. Bisphenol A-based applications are many and varied, ranging from DVDs, computers and home appliances to glasses and construction frames and lenses, paints, coatings for food and beverage cans. Small amounts of Bisphenol A are also used as components of formulations of antioxidant additives in soft PVC (wires and cables) and as a color preparer in thermal papers.
Octylphenois and Nonylphenols (NP): are organic compounds of the large family of alkylphenols. They are products of industrial synthesis formed during the alkylation process of phenols, particularly in the synthesis of polyethoxylated detergents. They are found in many industrial, domestic, agricultural, cosmetic and pharmaceutical products. They are mainly used as detergents, emulsifying agents, wetting agents, solubilizers and degreasing agents.
Water Contamination
Water bodies are one of the major sources of EDCs exposure. Surface and underground waters can be contaminated by industrial, agricultural and domestic effluents. The last one is one of the biggest sources of estrogen pollution. Both men and women daily secrete a number of estrogens through urine. Unfortunately, most urban sewage treatment stations are not effective on the removal of this compounds from water, allowing the continuous return of these contaminants to nature.
With the advances of the pharmaceutical companies, the use of hormonal contraceptives has increased in the female population. In Brazil, about 79% of women use a contraceptive method, being 35% of the hormonal kind. This has generated a greater release of EE2 in the environment.
Also, there has been a greater worldwide consume of several other products containing estrogens, such as other pharmaceutical products, cosmetics, pesticides, plastic packages, and others.
The effects of this increased consumption can be seen in the rivers and other aquatic environments. In Brazil, the concentration of estrogens in some cities is alarming.
Estrogens Dangers
Ecotoxicity
Once they are so biologically active, the presence of estrogens in the water can directly interfere and cause harmful effects in the life cycle of many living things. In this context, fish are the most investigated, since they can be affected by the lower concentrations of EDC, thus been used as indicators of environmental changes. Among the most serious consequences of estrogens is the decrease in growth and reproduction rates, related to the reduction of the reproductive system and even the feminization of the males. Both in fish and in amphibians and birds, these effects can be analyzed through biomarkers such as vitellogenin (VTG), a protein normally found only in females, whose production is stimulated with the presence of sex steroids.
Toxic effects on humans
There are no conclusive studies to prove the toxic effects of estrogens on human health through environmental exposures, which can be attributed to the marked difficulty in demonstrating a causal relationship when the process of endocrine disruption is addressed. Even so, it is known that there are numerous possible adverse effects on human health, especially related to changes in the endocrine system. These include effects on the female reproductive system - sexual differentiation, ovarian function, increased risk of breast and vaginal cancer, polycystic ovaries and endometriosis - and in the male reproductive system - reduced sperm production, increased risk of testicular cancer, and of prostate, infertility, and alterations in hormone levels of the thyroid.
One of the estrogens with the highest risk of toxicity is the Bisphenol-A (BPA) plasticizer. Therefore, due to a worldwide concern about the presence of this compound in plastic packaging, a report was organized by the United Nations (UN) in 2010:
“In light of uncertainties about the possibility of adverse human health effects at low doses of BPA, especially on reproduction, the nervous system and behavioural development, and considering the relatively higher exposure of very young children compared with adults, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) jointly organized an Expert Meeting to assess the safety of BPA.” (FAO; WHO, 2010, p. 7)
Even with inconclusive results, following this report and other studies from government organizations, several countries have now banned the manufacture of plastics containing Bisphenol-A. As well, a global demand for BPA-free products and packaging has started. In Brazil, the National Agency for Sanitary Surveillance (ANVISA) has banned, since 2012, the importation and manufacture of baby bottles containing Bisphenol A.
Existent Water Treatments
There is no legislation in force in Brazil regarding limit concentrations for endocrine disrupters in water, but it is still possible to see how alarming the concentrations found in water bodies are. Compared with the European Union proposed limit, which is 0.035 ng / L, most of the data found is well above this value and it is known that this contamination has a great possibility of worsening.
The major problem is that there is still no suitable method at Sewage and Water Treatment Stations that is fully effective in removing harmful amounts of estrogenic substances. In order to remove them, some processes are used:
Although advanced oxidation techniques are very efficient, they cause environmental problems that have already been reported and, because of their toxicity content, are harmful to human health. A more effective solution, which has been well studied, is the application of processes involving the use of enzymes capable of degrading EDCs substances. One of the most viable alternatives to obtain and apply such enzymes is by biotechnological means.
Our Sollution
Why Laccase?
Laccase is a polyphenol oxidase enzyme found in many organisms, from plants and insects to fungi and microorganisms. Known as a green catalyst, helps in the cleavage of aromatic compounds and, due to its low degree of substrate specificity, this protein is effective in the oxidation of various compounds.
The active site of the enzyme has a cluster of four copper atoms, which can be distinguished by spectroscopic analysis. The first atom consists of a blue copper ion (type 1), which classifies the laccases belonging to the family of the blue copper proteins. The other three atoms are divided into one of type 2 and two type 3 ions.
Source: BARRIOS-ESTRADA et al, 2018
The enzymatic effectiveness in the oxidation of phenols occurs due to the presence of this cluster in the molecule. While copper type 1 oxidizes the substrate, the other three atoms are responsible for reducing oxygen in water. In this reaction, the oxidation electron is transferred to the three copper ions, forming a trinuclear agglomerate, which transfers electrons to the terminal acceptor oxygen. Upon receiving four electrons, the oxygen has its complete reduction.
Source: CATHERINE et al, 2016
A variety of studies, in which laccase was used in the degradation of more varied micropolluantes from water and soil, was performed. In these, the enzyme demonstrated the potential of application in the remediation of several compounds, such as drugs, pesticides and the estrogens themselves.
Why Phoma sp. ?
Phoma sp. is a fungus appurtenant to the Ascomicota phylum, Pleosporales order, Pleosporaceae family and has more than 80 species. It’s possible to identify it by its spores found inside the mycelium fungal; this characteristic allows us to identify this fungus frequently. One of our two genes comes from Phoma sp. UHH 5-1-03 (DSM 2245):3073 pb – 607 aa. We choose this fungus enzyme because it copes with diverse physicochemical conditions found in aquatic habitat. Besides that, its laccase has a potential in degradation of endocrine-disrupting chemicals (EDCs), our chemical targets. Its studies have shown a promising biotechnological solution for EDCs degradation presents in water.
Why Pleurotus Ostreatus?
The genus Pleurotus, appurtenant to the basidiomycetes class, it’s composed by cosmopolitan, saprophytic or parasites species. This fungus can produce many enzymes, like peroxidases, laccases, cellulases, hemicellulases and xylanases, characteristic that allow it to grow in lignocellulosic substrates. The other laccase we took from Pleurotus ostreatus NRRL0366. We chose it because its protein has a good potential degrading phenolic compounds, like estrogens; its best activity is degrading 17α-ethinylestradiol, a synthetic hormone.
Why E. coli ?
Escherichia coli is a model organism, well-studied and widely used as a chassis. Its metabolism is already well known and the parameters to control it’s growing are also dated. The growing kinetics of E. coli is also a positive point, since it is fast, is also aligned to one of our purposes for the project, which is a scale-up production of laccase enzymes.
For cloning we selected the E. coli DH5-α strain, that is engineered to optimize the transformation. For the expression of our enzymes we chose E. coli BL21 cells, that are engineered to optimized and handle the expression of a protein of interest.
REFERENCES
ANVISA. Bisfenol A. Disponível em < http://portal.anvisa.gov.br/ alimentos/embalagens/bisfenol-a>. Acesso em11 abr 2018
AQUINO, S. F. DE; BRANDIT, E. M. F.; CHERNICHARO, C. A. DE L. Removal of pharmaceuticals and endocrine disrupters in sewage treatment plants : literature review Remoção de fármacos e desreguladores endócrinos em estações de tratamento de esgoto : revisão da literatura. Eng Sanit Ambient, v. 18, n. 3, p. 187–204, 2013.
BARRIOS-ESTRADA, Carlos et al. Emergent contaminants: Endocrine disruptors and their laccase-assisted degradation – A review. Science Of The Total Environment, [s.l.], v. 612, p.1516-1531, jan. 2018. Elsevier BV
BILA, D. M.; DEZOTTI, M. Desreguladores endócrinos no ambiente: efeitos e consequências. Quimica Nova, v. 30, n. 3, p. 651–666, 2007.
CATHERINE, Hautphenne; PENNINCKX, Michel; FRÉDÉRIC, Debaste. Product formation from phenolic compounds removal by laccases: A review. Environmental Technology & Innovation, [s.l.], v. 5, p.250-266, abr. 2016. Elsevier BV.
DIAS, Raissa Vitareli Assunção. AVALIAÇÃO DA OCORRÊNCIA DE MICROCONTAMINANTES EMERGENTES EM SISTEMAS DE ABASTECIMENTO DE ÁGUA E DA ATIVIDADE ESTROGÊNICA DO ESTINILESTRADIOL. 2014. 177 f. Tese (Doutorado) - Curso de Programa de Pós-graduação em Saneamento, Meio Ambiente e Recursos Hídricos, Universidade Federal de Minas Gerais, Belo Horizonte, 2014.
HISS, Haroldo. Cinética de Fermentações: Uma análise matemática da atividade microbiana. São Paulo: Ministério da Cultura, 2013. 654 p.
Joint FAO/WHO expert meeting to review toxicological and health aspects of bisphenol A: final report, including report of stakeholder meeting on bisphenol A, 1-5 November 2010, Ottawa, Canada.
KIDD, Karen A. et al. Collapse of a fish population after exposure to a synthetic estrogen. Pnas, Winnipeg, v. 104, n. 21, p.8897-8901, 22 maio 2007.
KIM, Y.-J., NICEL, J.A., 2006. Impact of reaction conditions on the laccase-catalyzed conversion of bisphenol A. Biores. Technol. 97 (12), 1431–1442.
LIBARD JUNIOR, N. Estudo de lacases fúngicas para degradação de compostos interferentes endócrinos. Dissertação de mestrado, UNIVILLE, 2010
LOPES, L. G. et al; ESTROGENS IN NATURAL AND TREATED WATERS IN THE REGION OF JABOTICABAL – SÃO PAULO, BRAZIL. São Paulo, 2010.
ONU. Métodos contraceptivos em 2015. Disponível em
REIS FILHO, R. W.; ARAÚJO, J. C.; VIEIRA, E. M. Sexual estrogenic hormones: bioactive contaminants. São Paulo, 2006.
US EPA (United States Environmental Protection Agency). Nutrient control design manual: state of technology review report. The Cadmus Group, Inc., 2009, 104 p.
US EPA. Special Report on Environmental Endocrine Disruption: An Effects Assessment and Analisys, Report No. EPA/630/R-96/012, Washington D.C, 1997.
ZANIN, G.; MORAIS, F. F. Enzimas como agentes biotecnológicos; Said, S.; Pietro, R. C. L. R., eds.; Legis Summa: Ribeirão Preto, 2004