Study improves method for detecting mercury in fish

Fabio Reynol/Agência FAPESP/DICYT Mercury, a potentially toxic metal that is capable of accumulating in the body, is being found in Amazonian rivers as residue from mining activities and, in some locations, occurring naturally. Its presence is affecting the aquatic fauna and could affect humans who consume fish that contain mercury.

Initiated in 2011, a project coordinated by the Biosciences Institute of the Universidade Estadual Paulista (Unesp) in Botucatu (SP) has used the tools of molecular biology to improve methods for detecting mercury in the most frequently consumed fish from the Madeira River basin in the Brazilian state of Rondônia.

Funded by a regular research grant from FAPESP, the project “Development of analytical methods for mercury metallomics studies in fish collected in the AHE JIRAU - Madeira River Basin area of influence” analyzed three species of fish among those most frequently consumed in the region: catfish (Brachyplatystoma rousseauxii), pacu (Mylossoma sp., Myleus sp.) and jaraqui (Semaprochilodus sp.).

That work continued to improve upon the techniques employed in another project, “Development of analytical methods for evaluation of Nile tilapia (Oreochromis niloticus) metalloproteins” another regular research grant from FAPESP, carried out from 2008 to 2010 with fish of this species found in the state of São Paulo.

This time, however, the study was limited to the fish collected in the area of influence of the Jirau hydroelectric power plant, which in 2010 experienced construction of a dam along a 120-km stretch of the Madeira River in Porto Velho (RO). “This type of construction alters the dynamic of the river and could potentially release mercury that had been inert at the bottom of the river, and that may now be absorbed by the aquatic biota,” explained chemist Pedro de Magalhães Padilha, professor at Unesp and the project’s coordinator.

According to Padilha, the study managed to optimize metal speciation methods by making use of a rather recent field known as metallomics. A combination of proteomics and metal detection techniques, metallomics aims to determine the distribution of metal and metalloid species and to clarify the physiological and functional characteristics of biomolecules whose structures contain metal ions.

Padilha said that there are two ways for a protein to carry metals. The first is when the metal is part of the protein molecule itself – such as hemoglobin, a metalloprotein that has iron atoms that transport oxygen.

The other method of transport is when the metal or metalloid binds to the protein through non-specific ligands, forming what is known as a metal-binding protein. It is from this last group that the research team chose proteins capable of acting as possible biomarkers for the presence of mercury in the fish.

“We have identified eight types of proteins and 16 isoforms as strong candidates for biomarkers,” reported the professor. Isoforms are different forms of the same protein, but they are encoded by different genes and present small differences in their peptide sequences. The definition of an effective biomarker will be made in a future stage of the project. In this part of the project, the researchers improved the methods for metalloproteomics study of mercury, according to the Unesp professor.

The research began in Rondônia with the capture of the fish, biometric testing and removal of the muscle and hepatic tissue for analysis. The samples were frozen in liquid nitrogen at –190°C and sent to Unesp. There, researchers at the Botucatu laboratories handled one of the most complicated parts of the work: extracting the proteins.

“The challenge was to extract the protein without destroying or altering its structure, even if that meant destroying the very tissue that contained it,” explained Padilha. They opted to use a simple method: maceration of the tissue in liquid nitrogen and ultrapure water.

The aqueous solution that was obtained from this process underwent two-dimensional electrophoresis followed by synchrotron radiation X-ray fluorescence, a step performed at the facilities of the National Synchrotron Light Laboratory (LNLS) in Campinas (SP).

The work involved performing a qualitative mapping, which determined the protein spots, or possible proteins, that contained mercury. The quantitative determination of mercury in protein spots was made through graphite furnace atomic absorption spectrometry and a series of complex stoichiometric calculations. One of the results of the research included the development of a new technology for determining amounts of mercury, which was published in the journal Food Chemistry in December 2013.

One of the project’s most important revelations was the special relationship between mercury and small proteins. The metal was mainly found in proteins that had low molecular weight, which would be the principal carriers and strongest candidates for biomarkers.

The cells of hepatic tissues, however, are particularly important to this study because in the presence of some metals, the liver produces what are known as metallothioneins, detoxification proteins that are directly related to the presence of metals in the body.

In the animals studied in the project, no amounts of mercury were found to be greater than or equal to 500 micrograms/kilogram of meat, the maximum limit provided by the World Health Organization (WHO) for this metal.

Nevertheless, Padilha warned about the long-term effects. “We have to consider the fact that when a metal bonds to a protein, it is because it has displaced some other essential element that the body may need,” he said.

In addition to this, the researcher mentioned that mercury and similar metals have properties that allow them to build up in the body, causing adverse effects to health when they reach high levels over time.

Mercury in breast milk

To date, the research project has produced four published scientific papers and the doctoral dissertation “Development of analytical methods for mercury metallomics studies in fish collected in the AHE JIRAU - Madeira River Basin area of influence,” written by Paula Martin de Moraes, who received an educational scholarship from FAPESP to pursue her doctorate.

The work also resulted in the master’s thesis “Mercury metalloproteomics studies in hepatic tissue samples of fish collected in the the AHE JIRAU - Madeira River Basin area of influence,” by biologist José Cavalcante Souza Vieira, also a FAPESP scholarship recipient. Both were advised by Padilha.

Felipe André dos Santos took part in the study by examining another type of sample for his doctoral dissertation. Instead of fish, Santos applied the metallomics techniques to breast milk collected among the riverine population of the Madeira River for the purpose of detecting possible traces of mercury and finding biomarkers for the metal.

The resulted in the thesis “Metallomics study of mercury in breast milk collected from the riverine population of the area of influence of the AHE Jirau – Madeira River Basin.” “The proposal was based on the hypothesis that because the riverine population consumed more fish from the river than the urban population, the riverine population would be more at risk from the mercury found in the fish,” explained Padilha, who also advised Santos.

As in the larger project, Santos’ research objective consisted of identifying the proteins responsible for the transport of mercury. The only difference was that the samples in this case consisted of breast milk. Initially, the student selected nursing mothers who had been contaminated by mercury. This stage involved analyzing the hair of the women who were nursing because hair has the ability to accumulate potentially toxic metals.

Then, breast milk samples from the contaminated group were analyzed. Santos obtained the proteome of each sample using two-dimensional electrophoresis. Then, after subjecting the material to other analytical techniques, he selected proteins in which the mercury was shown to be present. This work led to the identification of the lysozyme C protein as a possible biomarker of mercury.

The research project coordinated by Padilha included the participation of specialists from the Federal University of Rondônia (UNIR), the University of Brasília (UnB), the University of Campinas (Unicamp), the National Institute for Research in the Amazon (Inpa), the Pontifical Catholic University of Goiás (PUC-Goiás) and the Energia Sustentável do Brasil (ESBR), the consortium that manages the Jirau hydroelectric dam.