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The danger mapped out

Studies confirm the relationship of free radicals with a series of diseases and give a new direction to treatment

SÉRGIO VANIN / USPThe Pyrophorys divergens firefly, with lanterns (yellow spots) on the thorax that emit a green light when the insect walks on foliageSÉRGIO VANIN / USP

Until a short time ago, the action of free radicals had been under suspicion – those molecules or fragments of molecules with an insufficient number of electrons, mainly made up from oxygen. Now, a series of studies confirms that they are responsible for the appearance and the worsening of several problems, such as hereditary diseases, lead intoxication, hypertension, schizophrenia, bipolar disorder, diabetes and cancer.

The tip of this iceberg emerged in 1978, with cases of anencephaly – fetuses without a brain – and respiratory diseases that popped up in the Vila Parisi district, in Cubatão, by the São Paulo coast. Prompted by news that high levels of pollution could be causing these two problems, chemists Etelvino Bechara and Marina Medeiros, both from the University of São Paulo (USP), went there to investigate. Once they had overcome the difficulties, they collected blood from the inhabitants of the district and found high levels of antioxidant enzymes – it was a sign of the excess of free radicals, which can be harmful for the organism. This clue did not succeed in providing a direct explanation for anencephaly, still today of unknown origin, but it did put the researchers onto fertile terrain and elucidated the role of free radicals in a series of other illnesses.

Free radicals are indispensable for the formation of deoxyribonucleic acid (DNA), the carrier of the genetic code, and they act in the destruction of bacteria and tumors, for example. But they behave like villains when produced and used in an unbalanced way – in a condition known as oxidative stress -, as they start destroying cell membranes, DNA and enzymes, creating a favorable situation for neurodegenerative diseases to prosper, like Alzheimer’s disease and the process of cell death.

A few years before the Vila Parisi episode, Bechara got to know, at a lecture in São Paulo, French researcher Adolf Michelson, from the Institute of Physicochemical Biology, in Paris. Michelson had found that the rural population of France showed lower blood levels of an enzyme called superoxide dismutase, involved in the elimination of oxygen radicals, than the Parisians. In parallel, Paulo Naoum, a biologist from the São Paulo State University (Unesp) in São José do Rio Preto, had discovered, in the blood of Cubatão residents, an excess of metahemoglobin, one of the compounds formed when hemoglobin, a molecule that carries oxygen in the blood, reacts with sulfur oxides. “As there was a lot of sulfur oxide in the air, Naoum attributed the alterations to pollution of the environment, but he was unable to explain how it modified the quantity of enzymes in the blood of the people who lived in Vila Parisi”, says the chemist from USP.

Bechara joined together the ends of each study and showed that, in the case of Cubatão, it was the free radicals that caused oxidative stress, according to the first Brazilian article on the subject, published in 1983 in the Archives of Environmental Health. Afterwards, he discovered one of the compounds that form free radicals: 5-aminolevulinic acid (Ala). A precursor of the heme group – a brown-red pigment that is part of the structure of proteins like hemoglobin and myoglobin (an oxygen store) -, Ala reacts quickly with oxygen and produces a large quantity of free radicals.

His findings came to be integrated with phototherapy research into cancer of the mouth, skin and rectum, which now offers the alternative of applications of Ala directly into the tumor tissue, followed up by irradiation. They also formed the basis of a proposal for an antioxidant therapy, today at the experimental stage, for acute intermittent porphyria, a disease of a genetic origin associated with an excess of Ala. With a greater prevalence in Scandinavia and in Britain, acute intermittent porphyria has subtle characteristics: at the majority of times, the victims do not manifest the disease.

In the whole of their lives, they may have just one or two acute crises, with strong abdominal pains, muscular weakness, hallucinations and a characteristic sign: their urine darkens and takes on a brown, red color. They may also develop liver cancer, which becomes more frequent with the number of attacks, in particular among women. Porphyria is attributed to a deficiency is attributed to an enzyme called 5-aminolevulinic acid deaminase and to the accumulation of Ala in the blood, brain and liver.

From porphyria to schizophrenia
Bechara decided to study this kind of porphyria after getting to know another work by the Frenchman Michelson, which showed an intensification in the levels of free radicals in French autistic children and in victims of schizophrenia and bipolar disorders. The chemist tested the hypothesis that the excess of Ala is the cause of these mental disorders in a work carried out in collaboration with the physician Paulo Marchiori, from the Hospital and Clinics (HC), and with biochemist Dulcinéia Abdalla, both from USP. They assessed both patients with acute intermittent porphyria in an acute crisis, when they show neurological symptoms and hallucinations, and the victims of schizophrenia and bipolar illness, treated at the HC and the Juqueri Psychiatric Hospital. The blood tests showed an increase in the antioxidant enzymes, which try to hold back the excess of free radicals – just like the inhabitants of Vila Parisi.

Little by little, it was becoming clear that the radicals caused chemical damage to other radicals. When exposed to Ala, oxyhemoglobin transforms itself into a methemoglobin, ferritin (a protein that stores iron in the organism) releases iron ions, and the DNA molecule breaks up under the attack from the free radicals. Under the action of the excess of Ala, the mitochondria, one of the compartments of a cell, swells up and stops producing adenosine triphosphate (ATP), a compound that is essential for the production of energy.

Step by step came the proof of Bechara’s hypothesis that free radicals derived from Ala are linked to the molecular bases of porphyria. Later on, other evidence: studies carried out in conjunction with Paolo Di Mascio, from the Chemistry Institute at USP, showed that rats treated with Ala showed from five to six times more damaged bases of genetic material than a normal rodent, besides accumulating iron in the liver and in the brain. “This may be involved in the genesis of cancer of the liver, one characteristic of acute intermittent porphyria”, Bechara notes.

A short time ago, Bechara and his collaborators demonstrated that flavonoids, natural substances found in grapes and green tea, may cooperate with vitamin C in protecting the mitochondria against being attacked by peroxides, substances that generate free radicals. They also put forward the proposition that aminoacetone, a derivative of the threonine amino acid, worked like a source of free radicals and a mobilizer of iron in diabetes mellitus.

The research branched out once again in March, when Cassius Stevani, a former student of Bechara during post-doctoral studies and a researcher at the Osasco Learning Institute Foundation (Unifieo), discovered luminescent mushrooms in the Upper Ribeira State Tourist Park (Petar in the Portuguese acronym , in the south of the state of São Paulo). For some time, bioluminescence, a chemical process by means of which light is produced, has proved to be a good model for studying the action of free radicals. Stevani discovered five species as yet not described scientifically in Brazil, which live on trees or decomposing leaves. According to Stevani, the chemical methods for the production of light are not known yet: “It is something totally new”.

Secrets of the rainbow fireflies
Chemists and biologists from USP spent many nights in the cerrado (wooded savanna) of Mato Grosso turning over rotten tree trunks, gullies and anthills in search of different glowworms, which emit light that varies in color from an intense green to red. For Etelvino Bechara’s team, the research into the bioluminescence of these insects ran in parallel with the one into free radicals. The lines of work finally joined up when bioluminescence revealed itself as a biological model for studying the accumulation of free radicals in organisms, called oxidative stress: the production of light depends directly on oxygen, very scarce in the insides of the rotten tree trunks where the larvae of the luminescent insects live.

When studying fireflies larvae, Bechara’s team discovered that bioluminescence is an auxiliary phenomenon for the antioxidant enzymes, which protect the larvae from the excess of oxygen, the main source of free radicals. As the chemists from USP have demonstrated, the luminescent substance produced by fireflies, called luciferin, probably has an antioxidant role, besides emitting light. When it finds itself in an oxygen rich atmosphere, the insect increases its production of luciferase, an enzyme that speeds up the production of light, of luciferin itself, and of antioxidant enzymes. “This is a strong indication that, in glowworms, luciferin may work as a natural antioxidant against free radicals”, Bechara comments. At the same time, Belgian researchers have shown a similar behavior by coelenterazine, the luciferin of luminescent coelenterates (jellyfish), which also suffer from free radicals.

In the last few years, the biologists from the team of Cleide Costa, from the Zoology Museum at USP and from the Natural History Museum of Basel, in Switzerland, who took part in this group, described three new species of a very rare family of luminescent beetles, the phengodidae, amongst them the Phrixothrix viviani. This insect is a unique model for the chemical study of the relationship between the structure of luciferase and the color of bioluminescence, for emitting two different colors at the same time: red from the cephalic lantern and green from the abdominal lanterns (the lantern is the organ that emits the luminescent light).

From one of the most productive expeditions, which took place last year, the researchers brought back 700 larvae and adults of Pyrearinus termitilluminans. Bechara and Graham Timmins, from the Dartmouth Medical School, in the United States, demonstrated that the consumption of oxygen is directly associated with the emission of light in the larvae of this species.

Variable color
In another article, published in Insect Biochemistry, Bechara and Marcelo Barros, also from the Chemistry Institute, registered the variation in production of antioxidant enzymes in these larvae and came to the conclusion: enzymatic activity is associated not only with the production of light, but in particular to the moments of intense physical activity, like digging tunnels or looking for food.

And there are coordinated biochemical paths that make it possible to minimize the excess of free radicals resulting from the oxygenation of the tissues. They also observed an unprecedented phenomenon that occurs with Macrolampis omissa, a species of firefly that is distinguished by the size of its lantern, which takes up almost half the abdomen. Naturally yellow, the lantern turns orange, and a yellow light is emitted during the nuptial flight and mating, which takes place on swamp grass, at twilight. But, at night, they mate on dry ground – and the light emitted is a yellowish green.

The Project
Oxidative Stress Associated with Porphyrias Characterized by an Overload of5-Aminolevulinic Acid (nº 96/00154-0Modality Thematic project; Coordinator Etelvino José Henriques Bechara – Chemistry Institute at USP; Investment R$ 62,495.20 and US$ 216,148.80