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biochemistry

Medicines from the sea

Research into marine resources finds anti tumor substances in sponges and tunicates on the coast

JOSÉ CARLOS FREITAS ArchiveAscidians: substances that attack tumorsJOSÉ CARLOS FREITAS Archive

The country with the second longest coastline in the world, Brazil has a coast that stretches for almost 8,000 kilometers, and only loses out to Australia. It is a privileged position for the rational exploration of marine resources, which include obtaining substances with potential use in biological systems, such as those that have been studied in a thematic project on renewable resources on the São Paulo coast.

Researchers from various institutions, who concentrated on the study of poriferans (sponges) and tunicates, have discovered in one of these living organisms two substances with unprecedented pharmacological potential in tumors, for which they have already filed for patents. Found in an organism of a tunicate, the ascidian Didemnum granulatum, the substances granulatimide and isogranulatimide are only two of the unprecedented compounds that were isolated and identified.

New species
Before the study, there had been only 34 species of sponges identified in the area. Collections that the researchers made in the channel and along the coast of São Sebastião Island, increased this number to more than 140 species, almost a half of the close to 300 already registered for the Brazilian coastline. Of the sponges removed in a strip of around 50 kilometers in extension on the perimeter of the channel, ten of them were new to Science, among them the Aplysina caissara, also with anti-tumor potential.

To collect and study the sponges and tunicates, the biologist and taxonomist Eduardo Carlos Meduna Hadju dived more than 200 times along the coast of Ilhabela island. For almost two years, he practically lived in São Sebastião. Hadju, who is a researcher with the National Museum of the University of Brazil and at the beginning of the project had been assigned to the University of São Paulo (USP), received two or three times per year a visit from Dr. Roberto Gomes de Souza Berlinck, a post-doctorate researcher in the chemistry ofmarine animals, currently at the USP of São Carlos. With Hadju, who had surveyed the distribution of the species in the region, Berlinck selected the animals collected for the extraction of substances and sent the extracts to the biology laboratory of José Carlos de Freitas, the project’s coordinator, for pharmacological testing.

An anti-cancer patent
The extracts of the substances collected are produced in a very simple manner. The animal is placed in alcohol – methanol or ethanol –, and the substances which it contains are dissolved in this way. After the alcohol is evaporated, the extract is submitted to a preliminary pharmacological test carried out by Freitas’s team. He directs the Marine Biology Center (Cebimar) of USP. During his training abroad, Berlinck also submitted 40 extracts to a new test, one which had just been developed in Canada. Some extracts showed activity and that which demonstrated the best performance, or that is to say, the greatest activity at the lowest concentration level, was exactly that which had been removed from the ascidian Didemnum granulatum found along the coast of São Paulo and Paraná.

Together with Raymond J. Andersen, of the British Columbia University, in Canada, Dr. Berlinck discovered granulatimide and isogranulatimide, substances with unprecedented pharmacological action on tumors. In March of 1998, the Canadian university, on behalf of both, requested a patent for these substances and it is currently under evaluation.

The pharmacological test was done on human breast cancer cells and basically detected the interference of substances in the organization of the DNA (deoxyribonucleic acid, the carrier of the genetic code) during the process of cell division. In this process, the substances inhibit the so called check point G2. The cells carry out two stops to verify the integrity of the genetic material. The first stop, or check point G1, happens shortly after cell division, to verify if the genetic material was not damaged in the process.

Then, the cell has a certain life time, which in mammals is 24 hours, before it splits once more. During this period, each cell carries out a new biochemical check, check point G2. “Close to 50% of the cells of solid tumors,” explains Berlinck, “don’t have check point G1 active and this is one of the reasons for which they are, shall we say, abnormal. If you inhibit check point G2, as does granulatimide and isogranulatimide, there begins a high mortality rate of the cells during division, since the DNA is already considerably damaged and no longer serves to maintain the cell alive.”

The researchers linked mild radiotherapy with the new substances, so both can totally inhibit the functioning of the DNA and to guarantee the death of the cells. The radiation dosage was low and the result showed the selective action of granulatimide and of isogranulatimide.

Hospital infection
Among the unprecedented compounds isolated during the research are four alkaloids, haliclonacyclamine E, and Arenosclerins A, B and C, obtained from the sponge Arenosclera brasiliensis, which only exists in Brazil. These compounds demonstrate cytotoxic and antibiotic activities against microorganisms resistant to antibiotics, those that cause hospital infections.In another sponge, the Aplysina caissara, which also only exists here, the team detected a large quantity of thealkaloid aeroplysinine-1, which also possesses potential anti-tumor action. In the 60s, Italian researchers had already found the same substance in another sponge, which today is going through clinical testing on human beings. In the case that the results are positive, Brazil could carry out the sea cultivation of Aplysina caissara and become a supplier of this alkaloid.

For coordinator Freitas, the more that is known of the marine biodiversity, the greater the probability of discovering substances with pharmacological potential, because the chemical diversity is directly proportional to the biological diversity. Poriferans and tunicates are especially potential sources of bioactive compounds. The researcher emphasized that “these groups also have structural importance in various ecosystems and are one of the global priorities in the study of marine biodiversity.”

Present in AZT
Many substances have already been isolated throughout the world during research into marine organisms, but few are under clinical testing. The complete process, form the discovery of a substance until its arrival on the market requires close to 15 years, between studies, tests and the approval of a patent. Meanwhile, synthetic derivatives of spongouridine and of spongothymidine , substances isolated from sponges during the 50s, were placed on the market as anti-leukemia and anti-viral compounds and served at models for the development of AZT, the basic drug in the treatment of Aids.

In theory, any substance can be synthesized, but the synthesis may well take a long time, while in a cultivated and managed organism the biosynthesis is very quick. “”For this reason,” adds Freitas “there is the necessity for an interaction of chemists with marine biologists, who know in detail the life cycles of the animals and of the adequate environment for their rearing.”

Search and monitor
The coordinator emphasized that research with substances from the sea has two facets. “One is to carry out a biochemical investigation of natural products, to check out biological activities and to identify molecules as yet unknown to Science, even with unknown bioactivity.” “The other line,” he continues “is the monitoring of the effects of molecules already known, on the environment and the health of people.”

Freitas, who is also a researcher at the Center of Applied Toxicology, one of the Research, Innovation and Diffusion Centers (Cepids) qualified through FAPESP, has, for example, a biology student with the responsibility of monitoring the presence of toxins in fish and in shell fish consumed by beach fishermen and tourists on the coast of São Sebastião.One of the harmful substances is tetrodotoxin, identified in the 50s,and exists in any sea and is found in fish, algae, tunicates and other marine organisms. Tetrodotoxin has already killed many people in Japan, but today, thanks to monitoring, it is rare.

On the Brazilian coast, a fish that is rich in tetrodotoxin is the bandtail puffer or spotted puffer (Sphoeroides spengleri), hated by the fishermen both for robbing the fishhook bait and for its toxicity: digesting 50 grams of it is a death sentence. Through experience and tradition, the beach fishermen know that this fish must not be eaten in contrast with the smooth puffer, of a much larger size, whose fillets are found in the fishmongers.

Also the environmental phenomenon a of the red tide can lead toxins directly or indirectly to human beings. Brought on by the abundance of one of the small organisms of marine plankton, – sometimes one, sometimes another- , and often the red sea is caused by a toxic species. Filtering organisms, such as mussels and tunicates, end up concentrating the toxin, which causes problems such as diarrhea in those who eat them.

The majorities of red tides happen with the multiplication of some microscopic alga, but can also be provoked by animals. On the São Paulo coast there has already been one caused by the ciliate protozoa Mesodinium rubrum and another by the tunicate Weelia cilindrica.

Freitas, who was born in São Sebastião and is the son of a fisherman, intends to proceed with this research. There are lots and lots of other studies under development at USP to investigate new substances found in algae, anemones and other marine organisms. For this type of research, the work of the taxonomist such as Hadju is crucial. Through the project’s finances, he has created the web page Brazilian Poriferans (www.geocities.com/labpor), as well as a CD-ROM, in demonstration version, which identifies sponges in the São Sebastião channel.

Sponges and tunicates, filtering organisms

Known as sponges, the poriferans are the most primitive multi-cellular organisms that exist. They have a history of more than 1 billion years, and although they inhabit almost all aquatic environments, they predominate in the sea. Filtering animals, they have a porous body structure – hence the name poriferans given to their phylum – one of the divisions of taxonomy or the Science of the classification of living things. The sponges inhabit shallow or deep waters and fix themselves onto the sand, on mud or rocks. The large variation in the format and colors makes them sometimes be confused with algae or other organisms. Their size ranges from a few millimeters to a meter in height for some species. The colonies that they form can cover several meters of aquatic floor.

Some, such as the genre Spongia, found in Mexico and Japan, have their bodies sustained by spongin, a protein similar to collagen, made up in such as way that they become appropriate for use as bath sponges. Another group of sponges are not soft. They have their skeleton formed by spikes made from silica or calcium carbonate and there are also cases in which the skeleton is of dense limestone.

The biologist Eduardo Hadju explains that the sponges remove the nutrients necessary for their survival from organic particles transported by the aquatic currents. These particles circulate in the organism through a system of exclusive channels, the aquifer system. The researcher is working on the taxonomic revision of a sub-order of sponges of the class Demospongiae, a very abundant and poorly investigated group from the Brazilian coast.

Ascidians
The tunicates in general don’t have popular names with exceptions such as the pyuria which in Chile is collected for human consumption. They are marine invertebrates covered in a type of outside coat formed from tunicin, a substance similar to cellulose, and they make up the sub-phylum Tunicate or Urochordate. We are dealing with a primitive group of the phylum Chordate in which the typical distinction of the chordates, the dorsal nerve tube, is only present during the larva phase. On the evolutionary scale, the tunicates are closer to the vertebrates, which as well are a sub-phylum of the chordates.

Among the tunicates, the project concentrated mainly principally on the group of ascidians, found throughout the world, from the shallows between seas until depths of more than 8,000 meters, on their own or grouped in colonies. They also feed by the filtration of water and the majority live stuck to rocks, the sides of wrecks and on shells, or anchored to the sand or mud. Anyone looking for ascidians can find them from the size of a small mark on a rock to an animal of 10 centimeters in height and with two visible siphons, one which brings water into its body, along with food and oxygen, and the other which removes the water together with excretion products and carbon dioxide gas.

The Project
Renewable marine resources of São Paulo State coast. Porifera (Demospongiae) and Chordata (Tunicata): taxonomy, chemistry and farmacology (nº 96/04316-5); Modality Thematic project; Coordinator José Carlos de Freitas – BioSciences Institute andMarine Biology Center (Cebimar) of USP; Investment R$ 600,000.00

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