Biologists from Brasilia have managed to explain how reptiles and amphibians resist the brutal transformation which they undergo when coming out of hibernation, after living for several weeks frozen. It is a delicate moment. The animals thaw out in a few hours, accompanying the increase in the temperature, which goes up from several degrees below zero to about 20º Celsius (C). Oxygen starts circulating again in the body, but in high quantities, and generates reactive forms, the free radicals, harmful to the organism.
Marcelo Hermes-Lima and his team from the University of Brasilia (UnB) have discovered that the animals get themselves ready during hibernation, in order to reduce the damages caused by the momentary excess of free radicals. Even frozen, working at a slower pace, the organism of some species of toads, turtles, snakes, and mollusks produces and stores antioxidant enzymes, in particular catalase, superoxide dismutase, and glutathione peroxidase, which degrade compounds like hydrogen peroxide (H2O2), formed in abundance as a consequence of the rush of oxygen.
The research that explains the tolerance to extreme cold and the lack of oxygen are helping to perfect the techniques for conserving organs, which still have to be transplanted within a few hours from having been removed from the donor, to avoid the death of the tissues. Human beings undergo a situation similar to the situation of the toad that thaws out at the end of hibernation, when the circulation of the blood in the heart or the brain becomes obstructed momentarily. With the return of the oxygen, there is a high risk of free radicals arising in excess and grave damage during a heart attack or a stroke. “The human organism does everything possible to defend itself from the free radicals, but we cannot count on a response in advance, like other animals”, Hermes-Lima says.
Snow and desert
Reptiles, amphibians, and mollusks produce enzymes in advance, when they regularly undergo three kinds of extreme situations, according to Hermes-Lima. The first is connected with extreme cold, which leads the animals, particularly in the Northern Hemisphere, to hibernate, as a way of saving energy. The other is exaggerated heat, in so-called aestivation, or summer hibernation: faced by the lack of water, snails from the semi-arid lands of the north of Africa, including some edible species of escargot, hide in their shells, and remain there for up to two years, with the organism partially dried out, until the rainfall comes back.
Finally, some vertebrates, like the red-eared slider (Trachemys scripta elegans) or the northern leopard frog (Rana pipiens) face up to the scarcity or even complete lack of oxygen, along with the winter and hibernation in frozen lakes. Reptiles like alligators and even mammals like seals and sea lions undergo a more humdrum situation – the lack of oxygen in their muscles and in organs like the kidneys and liver – when they dive and stay up to an hour without breathing under water.
In response to the extreme temperatures and to the lack of oxygen, the organism begins to work at a slow pace, in a state known as metabolic depression. At these times, the synthesis of proteins, the burning of sugars, the frequency of the heartbeats, and the rhythm of respiration fall sharply: in an extreme case, the metabolism of the arctic ground squirrel (Spermophilus parryii) is kept at 5% and the consumption of oxygen at 2% of normal, during the winter. “Even though everything has almost been brought to a halt, the antioxidant enzymes are a priority and continue to be produced”, Hermes-Lima says. This capacity is a result of evolution: only the animals that have managed to stock up with enzymes capable of withstanding the flood of oxygen have survived.
“We have discovered a trend in nature”, says Hermes-Lima. His studies – carried out in conjunction with a group led by Kenneth Storey, a specialist in metabolic depression at Carleton University, showed that the enzyme produced in greatest intensity before the oxygen comes back is glutathione peroxidase. The conclusion is based on an analysis of the antioxidant defense mechanism developed by animals like the red-sided garter snake (Thamnophis sirtalis parietalis), the northern leopard frog and the wood frog (Rana sylvatica), the goldfish (Carassius auratus) and in two kinds of escargots, the terrestrial snails Otala lactea and Helix aspersa.
Sometimes, some enzymes are produced less intensively. In a study published last year in the Canadian Journal of Zoology, Hermes-Lima and his students Marcus Ferreira and Antonieta Alencastro show that in a species of freshwater snails, the Biomphalaria tenagophila, there is a lower production of the catalase enzyme when the animals stay without oxygen for 24 hours, at the bottom of a jar with water, and of superoxide dismutase, when they spend 15 days in aestivation, under a continuous temperature of 26 °C. In both cases, there was a small increase in the quantity of the glutathione peroxidase enzyme. “Nobody can explain very well why the production of some enzymes increases and of others falls”, Hermes-Lima says. “But one of them going up must be sufficient.”
Snakes on ice
Hermes-Lima started to become familiar with frozen animals at the end of 1990. It was when he visited Storey at his laboratory in Canada, months after having met him in São Paulo, and he proposed to him that they should look for antioxidant mechanisms associated with freezing and the lack of oxygen, something that the Canadian biologist had not yet studied. Storey liked the proposal. In the following year, the Brazilian researcher landed in Ottawa, the Canadian capital, for a stay of two and a half years.
He began by working with garter snakes, found almost all over the North America, up to the north of the Canadian provinces. They are the first reptiles to wake up after hibernation, at the end of the winter. Except that, when spring arrives, the rivers melt in a few days and often cover the holes where the snakes had been lodged. But they can remain up to two days without oxygen, before leaving their waterlogged lairs. They can also stand being frozen for several hours.
Hermes-Lima imagined that there would be an abundance of antioxidant enzymes in these animals, as a way of preventing damage from the excess of oxygen. But it was not what he discovered. The snakes had, actually, small quantities of antioxidant enzymes, when compared with rats, but these quantities would increase according to the situation. The glutathione peroxidase was predominant under a five hour freeze at 2.5 °C, while in the following experiment, with the snake kept without oxygen over ten hours at 5 °C, the enzyme found in greatest quantity was superoxide dismutase. To start with, Storey did not believe it.
“He would say – Too cold, it’s not possible – and spent months without giving me attention, thinking I had done something wrong”, Hermes-Lima recalls. When the experiment was redone, the same results emerged. Finally convinced, but still intrigued, thinking that every cell ought to have a lower quantity of these enzymes under these extreme conditions, Storey agreed to sign, with the Brazilian, the article with these discoveries, published in 1993 in the American Journal of Physiology.
Twenty minutes without air
Since 2001, the team from Brasilia has been studying stress from diving in animals from Brazilian fauna. In two expeditions to the Pantanal (swampy lands), the researchers collected samples of tissues of embryos of the yacare caiman alligator (Caiman yacare) and also of newly born, young, and adult animals. When they dive and stop breathing, these reptiles give priority to the circulation of oxygen, which becomes scarce. The blood ceases to go to the muscles and to such organs as the liver, and goes towards priority targets like the heart and the brain, so as to maximize the time they stay down under – up to 20 minutes.
Without the possibility of carrying out experiments on the caimans in the laboratory, since here was an animal somewhat larger than the usual mice, the team from the University of Brasilia built a map of the damage caused by the excess of free radicals in lipids (fats) and proteins in the course of the development of these reptiles. “The damage is greater in tissues with a higher metabolic rate, like the brain, liver, and kidneys”, Hermes-Lima explains. His team has also been working since last year with samples of skin and the fatty layer of humpback whales (Megaptera novaeangliae), which spend a part of the year along the south coast of Bahia and stay below the water, without breathing, for up to 20 minutes.
Molecular Physiology of Free Radicals in model Systems; Coordinator
Marcelo Hermes-Lima – UnB; Investment R$ 97,000.00 (CNPq)