Difference between revisions of "Team:USP-EEL-Brazil/Background"

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.

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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:

• Physical processes: membrane adsorption or filtration and ultra violet.

• Chemical processes: advanced oxidative processes (ozone or peroxides), absorption by coal.

• Biological processes: activated sludge and membrane bioreactors.

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.

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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.

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