This year’s project for the team Tec-GenetiX CCM consists on the bioremediation in aqueous media of molecules that are similar to estrogen, such as BPA, PCB, PBDE and phthalates that are common secondary products in the manufacture of many common plastics. These toxins are released in the solution in the presence of high temperatures (NIH-HHS, 2010 p.1) as well as alkaline conditions or when the plastic is past its date. Other factors such as mechanical stress, UV light, dyes and pigments, metallic contaminants and amine components will also increase degradation (aging is being researched) (Pedersen GA, Hvilsted S, Petersen JH, 2015 p.6). As their structure is similar to several hormones used by organism to maintain their endocrine system the substances are considered pollutants for the human body and the environment. However, there are no regulations in Mexico regarding these compounds and thus they represent a health threat for plastic products consumers.
What They Do
Nowadays, several substances are being studied by their toxicity in the human body and environment. Some of them are substances that are added to the most used material in the XXI century: the plastics. Examples of these compounds are bisphenol A (BPA), phthalates, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). These compounds have been categorized as substances that cause a disbalance in the homeostasis of the organisms, also called endocrine disruptors. “Endocrine disruptors are chemicals that may interfere with the body’s endocrine system and produce adverse developmental, reproductive, neurological, and immune effects in both humans and wildlife” (NIH, 2016).
In the case of the aforementioned compounds, they disturb the endocrine system in two different endocrine axes: the sexual and the thyroid axes. These compounds have chemical structures very similar to estrogen, and thus the body can confuse them with the real estrogen molecule and estrogen receptors may bind to that molecule. In the case of BPA, various scientific studies have shown its ability to mimic female hormones such as estrogen. This capability has been linked to health damage as changes in behavior, hyperactivity, diabetes and obesity, early puberty, reducing the amount of sperm, prostate disease and prostate cancer, cancer breast, chromosomal abnormalities, brain damage, impaired immune function, decreased of antioxidant enzyme levels and abnormalities in the male reproductive system during pregnancy. (Gonzales 2011, p.62) In the case of the phthalates, they can either mimic or block female hormones, or in males, suppress the hormones involved in male sexual development. Its exposure has been found to be associated with numerous reproductive health and developmental problems such as: early onset of puberty, interfering with the male reproductive tract development, interfering with the natural functioning of the hormone system, causing reproductive and genital defects, lower testosterone levels in adolescent males, lower sperm count in adult males (Zero Breast Cancer, 2007).
PCBs are a group of chemicals of 209 aromatic chlorinated hydrocarbons used for products ranging from fluorescent light fixtures to coolant fluids inside parts of consumer electronics. Its exposure can cause liver damage, affect the reproductive development of children of exposed mothers, leads to reproductive changes such as thickening of the endometrium (McGovern, 2006), In addition, PBDEs have a similar effect to the PCBs, but they can be passed in breast milk. There have been some reported human health effects such as reduced fertility in women have been documented, and since they act as endocrine disruptors due to the aforementioned similarity between the thyroid hormones and the compound, they can deteriorate the development of the nervous system (Madrigal, 2012). Studies done on mice suggest that continuous ingestion of food with this compound affect the liver and the thyroid, even though more studies have to be carried out to give a definite answer to the question regarding the extent of PBDE’s health effects (“ToxFAQs™: Polybrominated Biphenyls (PBBs),” 2004). Other invertigations sucha as the effect of PBDE-99 (flame-retardant chemical) in rat sexual developpment of Ernie Hood (2006) have shown pronounced decreases in circulating sex steroids (estradiol, testosterone), reduced male anogenital distance, slight acceleration in onset of puberty, feminization of male, reduced ovarian follicles, impaired reproductive life span, decrease in size of pituitary gland in males, and increase in size in females (Hood, 2006 ).
Furthermore, these chemicals have been studied as interrupters to the metabolic pathways of thyroid hormones. The mechanism of action consists of the interruption of the transport proteins responsible to conduct T3 and T4 into the bloodstream. This thyroid-disruption is related to have deleterious effects on neurological outcome in pregnant women and infants (Boas, Malene, Feldt-Rasmus, Ullasen, M. Main, Katharina. 2012 p.240). Even more, they have been suspected to impair development of the human central nervous system (ML, Jugan, Y, Levi, JP, Blondeau, 2010. p.939). The same effect has been studied in communities of animals, specially the freshwater and marine wildlife (Bergman A, Heindel J, Kasten T, Kidd K, Jobling , Neira M, et al. 2013. p. 98).
There is already concern regarding these compounds in other countries. In Europe, the EFSA (European Food Safety Authority) proposed the Tolerable Daily Intake (TDI for their acronym) 0.05 milligrams of bisphenol A per kilogram body mass per day (mg / kg.m.c. / day). This parameter is based on No Observed Adverse Effect Level (NOAEL) of 5 milligrams per kilogram of body mass per day studies with multiple generations of rats (Møller, Leck, Og, Bo, & amp; Dhi, 2013).
US EPA (Environmental Protection Agency [Protection Agency Environmental]) established in 1987 that a safe dose BPA exposure was 50 mg / kg / day. On the other hand, since 2011 in California standard Toxin-Free Infants and Toddlers Act was accepted which prohibits the use of BPA in infant bottles (Prada, 2013). Finally, the FDA (Food and Drugs Administration) made a legislation to amend the market in July 2013, which prohibited the use of 98 Bisphenol A in making baby bottles and cups (European Information Centre on Bisphenol A, 2014). The legal context in Latin America is very little. The Mercosur as a "positive list polymers and resins for plastic containers and equipment in contact with food "sets allowable limit migration of BPA 3 milligrams per kilogram of body mass.
Furthermore, the Mexican government only has legislation on the tightness and quality of the glass resins, varnishes and canned, but not over limits compounds such resins (Gonzalez Castro, 2006). Currently, only Colombia has banned plastic processing with BPA that come into contact with food (European Information Centre on Bisphenol A,2014).
How We Get There
We were investigating a way to detect BPA and phtalates in contaminated water by a biosensor connected to a chromoprotein. This idea would give an specific way to detect estrogen-like activity of the substances and detect the amount of released compounds in the plastic water bottles. When the investigation was proceeding, we arrived to the investigation of Marchesini GR, et at (2008). This paper talks about a putative biosensor tested for 62 chemicals including the natural hormones (estrogen, progesterone, testosterone), polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals. The investigation showed an affinity of the compounds between moderate to high affinity for TBG.The highest affinity was observed in BDEs. On the other hand, TTR showed affinity to PCBs and PBDEs, halogenated BPA and genistein (Marchesini et al., 2008).
Figure 1. How endocrine disruptors act in the human body, affecting hormonal concentrations and having negative effects in different parts of the body (EDC report Ch2 2.5, 2013).
We decided that it was socially significant to get rid of these compounds in drinking water that comes from plastic bottles because Mexico is the biggest consumer of bottled water per capita (Medina & Smith, 2009). This was the way our project came into being.
To complement our project, we tend to use the 2015 of the team Carnegie Mellon, which used YFP for the bacteria to glow when estrogen was present in the solution. This would give us the opportunity to detect the substances and to give a report of the status of the water. If this two systems are linked, this opens the possibility of both monitoring and cleaning the water.
In addition, we are looking for the DNA basis of the Fusarium culmorum. It is a fungus that can degrade the plastic polymers by producing cutinases. In order to achieve this goal, we are looking for some collaboration from the mexican researcher Carmen Sanchéz from the National Council of Science and Technology (CONACYT).
About Our Project
Our project consist of transforming a non pathogenic strain of the bacteria Escherichia coli with plasmids with the genes of two of the human transport proteins Transthyretin (TTR) and Thyroxine-binding globulin (TBG). These molecules help iodothyronines to be transported in the human bloodstream, as they have a limited solubility in the blood serum. this carrier proteins have different affinities to the thyroid hormones. These proteins make a noncovalent association to travel to through the body. The most important one, is the Thyroid Binding Globulin (TBG) a single polypeptide chain of 415 amino acids which has a molecular weight of 63,000 daltons, as it is the major thyroid hormone transport protein in human serum with the highest association constant (Ka), with a Ka for T4 of 1010 M and for T3 of 109 M (Tetuya Shirotani et al., 1992; DeRuiter, J., 2001). It belongs to the serpin family in genomics and the specific gene for its transcription is located on the sex chromosome X at the locus Xq22.3, but the protein has no inhibitory function like many other members of the serpin family (NCBI, 2016). It is very stable although it loses its binding properties by denaturation at temperatures over 55°C and pH below 4. “Optimal binding activity requires the presence of the L-alanine side chain, an unsubstituted 4′-hydroxyl group, a diphenyl ether bridge, and halogen (I or Br) constituents at the 3,5,3′ and 5′ positions” (Samuel Refetoff, M.D., 2015).
Figure 1: 4X30 Crystal structure of human Thyroxine-binding globulin complexed with thyroine at 1.55 Angstrom resolution. (Bank, R. P. D., & Zhou, 2006).
The second transport protein is the transthyretin (TTR),a protein constituted of a tetramer of identical subunits, with 127 amino acids in each of them. (Samuel Refetoff, M.D., 2015). It transports the thyroid hormone thyroxine (T4) in the plasma and cerebrospinal fluid, and also transports retinol (vitamin A) in the plasma. The location of this gene is in the chromosome 18, in the locus 18q12.1 (“TTR transthyretin [ Homo sapiens (human) ],” 2016). This transport protein has a lower accessibility coefficient than TBG with Ka for T4 107 and for T3 of 106, but the expression is higher in the human with 25 mg/dl compared to the 1.5 mg/dl of the TBG (DeRuiter, J., 2001).
Figure 2: 5CNH X-ray structure of perdeuterated wild-type TTR at 1.42A resolution (Moulin & Erba, 2016)
Why Does It Work?
Figure 1. Structures of T4 , T3 , rT3 and T2 thyroid hormones. These structures share similarities with the compounds we are trying to bioremediate. Retrieved from http://ro.uow.edu.au/cgi/viewcontent.cgi?article=1029&context=scipapers (Hulbert, 2016).
The thyroid hormones T3 and T4 posses a similar structure to the toxins we deal with. They possess a double ring structure joined by an oxygen, forming a diphenyl ether structure, as can be seen in Figure 1. In the case T4 the outer aromatic ring has a hydroxyl group and two iodine groups in orto positions. The inner rings have two iodines in orto position, but it also has an amine and carboxylic group in the para position. In the case of T3, the structure is really similar, but instead of having to iodines in orto positions, it only has one (McCurry, 2010). These structures are really similar to that of BPA, PBDEs and PCBs because they all have aromatic rings and oxygen groups, and in the case of the PCB and PBDEs it also has a halide group of chlorine and bromine respectively, as Figures 2, 3, 4 and 5 show.
Figure 2. BPA’s skeletal and molecular structure. It shares the double aromatic ring with thyroid hormones, as well as the hydroxyl group at one side. This similarity will allow it to bind to the protein of our choice. Image retrieved from: Pubchem. Bisphenol A (CH3)2C(C6H4OH)2. https://pubchem.ncbi.nlm.nih.gov/compound/Bisphenol_A#section=3D-Conformer
BPA has the same structure of two benzenes but it's smaller in size than the thyroid hormones because there is no R group in para position in one of the rings. This characteristic gives the molecule a higher affinity to the transport protein and a higher period of permanence in the active site of the protein, since there are two equally viable binding sites in the hydroxyl groups. The main difference with the thyroid hormones is the absence of an R chain in para position and orto positioned halide groups.
Figure 5. Phthalate skeletal and molecular structure. It has several oxygen groups and an aromatic ring of benzene. Pubchem. Phthalate C8H4O4-2. Retrieved October 18, 2016, from https://pubchem.ncbi.nlm.nih.gov/compound/phthalate#section=3D-Conformer
Phthalates, however, have a lower capacity to bind to the transport protein TTR but not in the case of TBG that binds to T3 which has an R group. Phthalates are the least similar to the thyroid hormones, but the presence of an aromatic ring and oxygens with lone pairs of electrons allow the compounds to bind to the proteins.
Figure 4. PBDE molecular and skeletal structure. The ether group is similar to that of the T3 and T4 , , which makes it bound more easily to the proteins we will be using. Pubchem. BDE no. 181. Retrieved October 18, 2016, from https://pubchem.ncbi.nlm.nih.gov/compound/16212144#section=3D-Conformer
PBDE, is the most similar compound of the four studied, since not only does it have halide groups, but also has an oxygen between the two groups, forming a diphenyl ether with halide groups. Thus, its affinity to both carrier proteins is higher since it has more similar functional groups, only missing the amide group and carbonyl group.