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Water Analysis

Luminescent yeast

Researchers use microorganism to detect hormone in rivers

Leo RamosCotia River, in São Paulo’s Metropolitan Region, has the highest levels of contaminationLeo Ramos

A new type of test conducted by a team of researchers from the State University of Campinas (Unicamp), in partnership with American colleagues from the University of Tennessee, revealed that some water sources in São Paulo State contain high levels of estrogen compounds, a class of contaminants that can cause serious damage to the environment, animals and human health. The test, based on transgenic luminescent yeasts, showed that this situation is most hazardous in the Cotia River, which runs through São Paulo’s Metropolitan Region and supplies water to several towns along the way. The research team tested water from four sources – namely, the Cotia, Atibaia and Sorocaba Rivers and the Tanque Grande reservoir, located near the town of Guarulhos. The research team also included technicians from São Paulo State Environmental Company (Cetesb), linked to the State’s Secretary of the Environment.

The analysis showed that 83% of the samples contained estrogen; 8 of the 16 compounds tracked by the team were found in at least one sample. “Our research showed higher levels in places widely known to be significantly contaminated by domestic sewage,” says toxicologist Gisela Umbuzeiro, a professor at the School of Technology of Unicamp, who coordinated the project. “This is not an alarming situation yet, but it demands immediate action by the authorities in order to improve sewage treatment in Brazil.” According to Gisela, the bioassay is highly sensitive and can be immediately applied by environmental authorities to monitor the quality of the water. “The project was created with this objective,” she says.

Estrogen compounds, also referred to as endocrine disruptors because they interfere in the hormone-producing endocrine glands, have raised concern among scientists and sanitation authorities because of their speedy dissemination in the planet’s water reserves. Studies have shown that they can provoke the feminization of fish and amphibians, generate sexual anomalies in mollusks and reduce the fertility rate of the Northern Hemisphere’s polar bears.

There is growing suspicion that the existence of these pollutants in the water consumed by human beings is anticipating girls’ first menstruation and reducing men’s sperm counts. These compounds are comprised of a broad group of hormones, among which are the natural female hormones estrone (E1), 17ß-estradiol (E2) and estriol (E3), the synthetic estrogen 17-alfa-ethinylestradiol (EE2), used in contraceptive pills, and bisphenol A, an industrial compound that is found in such products as polycarbonate plastics, epoxy resins, fungicides, and certain special paper.

The problem is that many of these substances are eliminated in urine. They also become contaminants when medical drugs are poured down toilets, from urine of animals that are given hormones, and from rain water that takes these contaminants directly into sewage systems or into rivers and lakes. The paint that covers the hulls of ships and boats is another source. The compounds come from different sources; if these compounds are not removed from the sewage systems, they end up contaminating rivers, lakes and water reservoirs. “Conventional water and sewage treatment methods are generally unable to remove these compounds very efficiently. As a result, they can contaminate the water used for human consumption,” says professor Wilson Jardim, head of the Environmental Chemistry Institute of the Chemistry Institute of Unicamp, and coordinator of a project on this matter. The project is sponsored by FAPESP.

Efficient existing sewage treatment methods – such as advanced oxidation and ultra filtration – can eliminate these hormones. These methods are not widely used, however, because they are very costly. The risk of contamination is higher in countries such as Brazil, which has inefficient sanitation services and many places where sewage treatment is non-existent.

The importance of the study conducted in Brazil is the fact that it is a pioneering study. After having developed the bioluminescent yeast, the researchers from the University of Tennessee started to use it in tests to detect estrogen activity in U.S. rivers such as the Potomac, which runs through the nation’s capital city. The results, however, were negative, because the existence of these compounds in the water sources was insignificant. “The innovative aspect of our work was that for the first time a wide-scale test using yeasts was applied to monitor the quality of untreated and treated water, and to compare the results with the chemical analyses of the estrogenic compounds. This kind of testing had never been conducted before,” says Gisela, who worked for 22 years at the Toxicology, Genotoxicology and Environmental Microbiology Department of Cetesb. The company asked the National Technical Biosafety Commission (CTNBio) for permission to use transgenic yeast in Brazil.

069-071_Levedura_193Tiago CirilloFuture monitoring
In view of the project’s good results, the company has advised that it intends to use the test for its monitoring activities. “We are adjusting the methodology and we are interested in implementing the test in our future monitoring activities of surface and underground water,” says Rúbia Kuno, currently the head of the same department at Cetesb.

The Blyes (acronym for bioluminescent yeast estrogen screen) test – used to detect estrogen – as well as the yeast used to monitor the quality of the water sources in São Paulo were developed by a team headed by microbiologist John Sanseverino and by molecular biologist Melanie Eldridge, from the Environmental Biotechnology Center at the University of Tennessee, with whom the researcher Gisela has maintained contact since 2008. “I visited the lab twice to learn how to conduct the test. Ana Marcela Bergamasco, one of my students enrolled in the Master’s Program at the School of Pharmacy at USP, went there as well,” says Gisela. In 2010, various samples of water were analyzed in the United States. In the beginning of 2011, Cetesb was able to implement the test with a batch of yeasts donated by the researchers from the University of Tennessee.

To develop the test, the American scientists had to initially alter the genetic code of the Saccharomyces cerevisiae yeast, in order to make it luminescent and capable of detecting the estrogen compounds. In the lab, the yeast received a gene that produces the human estrogen receptor, along with a set of genes that produce light; these genes came from a luminescent bacteria. “The production of the luminescent yeast strain took two years. It was ready in 2005 and is being continuously improved,” says Melanie Eldridge.

The gene that produces the estrogen receptor is always active in yeast; that is, it produces the receptor continuously. When the receptor binds with the estrogen compounds the tested water samples, the receptor plus the estrogen compounds activate the light-producing genes and the yeast becomes luminescent. To quantify this luminescence – which is not visible to the naked eye – the scientists use a very sensitive type of equipment called illuminometer. “The intensity of the light is in proportion to the quantity of detected estrogen compounds,” Gisela explains. This light measurement is always in proportion to a standard compound, such as the E2. “This is why we say that a sample has the equivalent of x nanograms of E2 per liter.”

Approximately 400 water samples collected from the Sorocaba, Cotia and Atibaia Rivers and from the Tanque Grande Reservoir were analyzed by Cetesb and Unicamp, with Melanie’s support. The American biologist spent three months in Brazil in early 2011 to help implement the test and to train the research team. The samples were collected, placed in ice-filled Styrofoam boxes and taken to the laboratory. At the laboratory, the samples went through a solid phase extraction process, the objective of which was to put them into a more concentrated consistence. This step is important because it facilitates the detection of estrogen compounds that might exist in the water.

The same samples were submitted to liquid chromatography and mass spectrometry to undergo chemical analysis. This chemical analysis method is already being used by several sanitation companies to detect estrogen compounds in water. The researchers then compared the results of the Blyes test with the results of the conventional chemical analysis.

“We concluded that our test is more efficient, easier to use and cheaper. Once it is implemented, it only requires a few low-cost reagents to prepare the yeast culture medium. The initial investment for the purchase of a light meter is less than 10% of the value of a liquid chromatographer, the price of which ranges from US$ 200 thousand to US$ 500 thousand. In addition, this method of testing is faster, because hundreds of samples can be tested each month. Once the method is standardized, it no longer requires a lot of infra structure to be operational,” says Gisela. Another advantage of the Blyes test is its sensitivity. In the case of the EE2, a very powerful compound, the quantification limit of the chemical method was estimated at 4.2 nanograms per liter (ng/liter), while the test with the yeasts is able to detect concentrations of no more than 0.01 ng/liter. In the case of the E2, the quantification limit per chemical analysis corresponds to 1.8 ng/liter in comparison with the 0.1 ng/liter of the Blyes method. This system does not substitute chemical analyses; however, it can be a highly efficient option in monitoring programs.

The researchers’ expectation is that these results will attract the attention of regulatory bodies linked to the environment and basic sanitation to establish rules and take corrective actions in the management of water supply and sewage services. “Our objective is to provide a contamination scenario of major water sources in the state and the tools for the solution or mitigation of the problem. We have to change the legislation to deal with this issue,” points out professor Wilson Jardim. In the future, bioassays with luminescent yeasts will probably be available to any interested party. “We are planning to market the Blyes test,” says Melanie. We are checking on how to sell the strains so that many more people – and not only scientists – can use them to test their water samples.”

The Projects
1. Occurrence and estrogenicity of endocrine disruptors in drinking water and water sources in São Paulo State (nº 2007/58449-2); Modality Thematic Project; Coordinator Wilson de Figueiredo Jardim – Unicamp; Investment R$ 349,285.93 and US$ 350,654.38 (FAPESP)
2. Implementation of test for the evaluation of endocrine activity in luminescent yeasts Blyes and Blyas (nº 2010/17918-2); Modality Regular Funding for Research – Visiting Researcher; Coordinator Gisela Umbuzeiro – Unicamp; Investment R$ 20,697.01 (FAPESP)

Scientific articles
BERGAMASCO, A.M.D.D. et. al. Bioluminescent yeast estrogen assay (Blyes) as a sensitive tool to monitor surface and drinking water for estrogenicity. Journal of Environmental Monitoring. v. 13, p. 3288-93. 2011.
MONTAGNER, C. C.; JARDIM, W. F. Spatial and Seasonal Variations of Pharmaceuticals and Endocrine Disruptors in the Atibaia River, São Paulo State (Brazil). Journal of the Brazilian Chemical Society. v. 22, n. 8, p. 1.452-62. 2011.