Brazilian group develops methods to detect veterinary drug residue in fish | AGÊNCIA FAPESP

A project coordinated by researcher Felix Guillermo Reyes Reyes is conducted under the auspices of an agreement between FAPESP and Agilent Technologies (photo: Neuza Campelo/Embrapa Amazônia Ocidental)

Brazilian group develops methods to detect veterinary drug residue in fish

January 08, 2014

By Karina Toledo

Agência FAPESP – Developing methods to detect veterinary drugs in fish, which can be useful for health surveillance, is the objective of a group of researchers coordinated by Felix Guillermo Reyes Reyes, a faculty member at Universidade Estadual de Campinas’s School of Food Engineering (FEA/Unicamp).

The project, which also involves researchers from Unicamp’s Chemistry Institute, Universidade Estadual Paulista (Unesp) and the Brazilian Agricultural Research Corporation, was one of those approved in the call for proposals launched in April 2013 by FAPESP and Agilent Technologies.

“We will study how veterinary drugs are absorbed and metabolized by three species of fish of high commercial value in Brazil: tilapia, tambaqui (Colossoma macropomum) and pacu. We will conduct residue depletion studies, particularly in fish filets, which are the parts most often consumed, to determine how long it takes for the administered drugs to fall below the maximum permitted level and to pose no human risk. That way, we will be able to establish the waiting period between the last application of a medicine and the time at which a fish is slaughtered for consumption,” explained Reyes.

“We will develop both analytical methods for the detection of specific molecules and multiresidue methods. From a health surveillance point of view, one of the objectives is to evaluate the use of substances not approved for fish growing.

“Today, there are currently two antimicrobials registered in the country for use in pisciculture – probably due to a lack of interest among veterinary medicine companies in licensing their products for use in the sector. There is, on the other hand, a strong suspicion that fish farmers are utilizing products registered for other species of animals because fish raised in these conditions are under major stress and are therefore very susceptible to infections. But this illegal use occurs without any research to verify the appropriate dose and without risk assessment,” said Reyes.

In addition to threatening the health of consumers, warned Reyes, the uncontrolled use of veterinary drugs in pisciculture could cause environmental problems and contribute to the further development of drug-resistant bacteria found today.

“Some of these substances were studied in other countries for use in pisciculture, but the results are not necessarily valid for Brazil because the environmental conditions are different, as are the species studied. All of this influences the way in which a drug is metabolized and, consequently, the waiting period,” said Reyes.

The Unicamp professor presented the details of the project at a symposium organized by FAPESP and Agilent on October 30. There, Reyes affirmed that Brazil, which has 12% of the fresh water available on the planet, has the right conditions to be a major exporter of fish and derivatives.

“For the federal government, aquaculture is an important activity for agribusiness. The FAO [Food and Agriculture Organization] believes that Brazil will be the main country producing fish that will help to feed the global population over the next 20 years. But we need to be careful not to pollute the environment in this undertaking. Scientific research is fundamental to guaranteeing the quality of production and the integrity of the environment,” he said.

Gaucher disease

Another project approved in the FAPESP-Agilent call for proposals came from Professor Aparecida Maria Fontes, a faculty member at Universidade de São Paulo’s Medical School in Ribeirão Preto (FMRP-USP). The objective is to develop a new medicine to treat Gaucher disease.

Classified as an innate error of metabolism, this genetic disease is characterized by deficiency in the production of a beta-glycosidase enzyme, also known as glucocerebrosidase. This enzyme is involved in the metabolism of lipids, more specifically glucocerebroside, inside cells.

In carriers of Gaucher disease, the quantity of the enzyme is insufficient to break down glucocerebroside at the ideal rate, so this lipid accumulates in the liposomes of cells, mainly affecting the liver, spleen and bone marrow, but also compromising other organs and systems, such as the central nervous system (in types II and III of the disease). In addition to increasing the abdominal circumference due to swelling of the spleen and liver, the symptoms include anemia, thrombocytopenia (a reduction in the number of platelets in the blood) and bone pain.

“There are currently four commercial proteins used in enzymatic replacement therapy for Gaucher disease carriers. Our objective is to develop an optimized platform to produce the beta-glucosidase using a synthetic biology tool. The project has some innovative features that will increase productivity and make the product cheaper,” explained Fontes.

The biodrugs used in enzymatic replacement therapy for lysosome dysfunction disease, explained the researcher, are produced by different cell lines and require several modifications for delivery to the target tissues compromised by the disease.

With the help of viral vectors, the group, coordinated by Fontes – which includes genetic medicine researchers from FMRP-USP’s Genetics Department and the Surgery and Anatomy Department for Domestic and Wild Animals at the USP School of Veterinary Medicine and Zoology – intends to develop a human cell line capable of producing much higher levels of the enzyme.

“We will use a virus that is incapable of replicating in the cellular medium but that contains elements that will recombine and integrate into the genome of the host cell. As a result, the cell will permanently express the gene of interest. But instead of utilizing recombinant DNA technology, we will use synthetic biology techniques to develop the genome of our expression vehicle, which is the virus,” commented Fontes.

According to the researcher, the platform developed for production of the enzyme could later be developed to adapt useful biodrugs for the treatment of other genetic diseases that affect metabolism. Several currently lack therapeutic options, for example Niemann-Pick disease, mucopolysaccharidosis type IVA and α-mannosidosis.

Metabolic map

Paulo Mazzafera, a researcher at Unicamp’s Biology Institute, also participated in the FAPESP-Agilent event and is the coordinator of a project approved in the first call for proposals, launched in 2011.

The objective of Mazzafera and his colleagues’ project is to discover how variation in temperature and in the concentration of carbon dioxide (CO2) in the atmosphere influences the synthesis of lignin (a structural substance in plants) in two species of eucalyptus: Eucalyptus globulus, which is native to cold regions and offers a greater yield of pulp; and Eucalyptus grandis, a species that is common in Brazil and has a type of lignin that is more difficult to isolate pulp from.

“The idea is to understand how the metabolism of these species works and, based on this information, to think about manipulating certain genes to alter the lignin pathways in the plant,” said Mazzafera.

In the project’s first year, the researchers measured physiological parameters such as growth, photosynthesis and sugar production in the two species at temperatures ranging from 5°C to 35°C.

Three temperatures were then used in the main experiment: 10°C, 25°C and 35°C. For each temperature, the scientists simulated two concentrations of CO2: 380 particles per million (ppm), which is the average concentration in Earth’s atmosphere; and 700 ppm, the atmospheric concentration forecast by the Intergovermental Panel on Climate Change (IPCC) for the coming years.

“The plants were grown for approximately 30 days under these conditions. We have collected the materials, and now we are beginning metabolomic analysis, or evaluation of the varied metabolites that the plant produces. We will also conduct a transcriptomic analysis, which consists of a database of messenger RNAs that allows us to see what is expressed the least or the most in each situation. In our case, the focus is lignin synthesis, but the database will certainly be rich enough to be used to study several other metabolic responses to the conditions in which the plant grows,” he explained.

To conduct the analysis, explained Mazzafera, Agilent made software, known as GeneSpring, available: this program is capable of integrating metabolomic and transcriptomic data and generating a metabolic map of the plant.

Partnership in research

At the opening of the symposium, the president of FAPESP’s Board of Trustees, José Arana Varela, highlighted the importance of bringing together researchers whose projects were approved in the two calls for proposals, to promote the sharing of experiences.

“This program began in 2011 and has been very successful. This is an opportunity to do an assessment and see if there are things that can be improved,” said Varela.

Jack Wonstrand, Director of University Relations at Agilent, stressed that the company has a strong commitment to academics in Brazil. “No place in the country is more important to us than São Paulo State. We are concentrating on building our academic relationships here, and our experience working with FAPESP has been excellent,” he affirmed.

The total number of resources available to support the proposals selected in the second call for proposals is equivalent to US$ 800,000, equally split between FAPESP and Agilent.




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