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toxicology

Altered functions

Experiments with animals attest to the possibility of changes in sexual behavior that may occur during gestation as an effect of pesticides and antiallergic drugs

EDUARDO CESARThe mice on the facing page look like males. They just seem to be. Although they have the reproductive organs of the masculine sex, they are different. They are the size of female mice, and, as they produce less masculine sexual hormone, they are less aggressive and less sexually active. If they are given estrogen, a feminine sexual hormone, after being castrated, they behave like females: when put into contact with another male, they bend down their back and offer themselves for copulation. The changes in sexual behavior manifest themselves only in the adult rodent, but the phenomena that are unleashed occur much earlier, between the end of gestation and the first days of life.

The cause of these alterations – known as demasculinization and feminization of offspring – is the exposure of pregnant mice to very low doses, which fall short of being toxic, of pesticides that are much used in agriculture, cattle raising and even in domestic pest control: phenvalerate and deltametrine, substances classified as type II pyrethroids, harmful to the central nervous system. Also, the consumption of water contaminated with lead or with medicines to combat allergies (antihistamines) can alter the sexual behavior of mice, as it has been proved by a series of experiments carried out by the team of researcher Maria Martha Bernardi, of the School of Veterinary Medicine and Zootechny at the University of São Paulo (USP).

Although they use rodents, the experiments serve as indicators of problems that can affect the reproduction of domestic and wild animals, and, in extreme cases, of human beings, although the dosage of the medicines given to the rodents is from three to ten times higher. The results stress the need for care in applying insecticides in plantations that expose workers and pregnant women to risks – in particular between the sixth and ninth month of pregnancy, when the formation of the region of the brain associated with sexual behavior is completed: the hypothalamus. According to the National Toxico-Pharmacological Information System (Sinitox in the Portuguese acronym), in 2000, agro-chemicals were the fourth largest cause of intoxication in the country: there were 5,127 cases of poisoning and 1,378 intoxication, corresponding to 29% of all work-related contamination.

Limited risks
Studies are calling attention to the risks brought about by two antiallergics used to treat rhinitis (irritation of the nasal mucus) and conjunctivitis (irritation of the membrane of the eyelid): diphenhydramine, still used against sickness in pregnancy, and astemizole, prohibited last year for affecting heartbeats. They also warn of the dangers from the contamination of the environment by lead, a toxic metal released by burning fuel and by industries, which builds up in the organism.

Maria Martha believes that the same effects observed in animals, in the laboratory, may happen to humans. “Pregnant women should avoid contact with these substances, which can go through the placenta and reach the baby’s brain”, she recommends. “Should this occur, the effects would only be perceived at puberty”. But, although possible, the risks are rather improbable, since it is not always that a substance that has not proved to be safe for mice is harmful to humans – the reverse is true. “Research with animals uses much higher doses than the therapeutic ones”, explains Anthony Wong, the director of the Center for Toxicological Assistance of the Hospital and Clinics of USP’s School of Medicine. “Mice are part of the first group of mammals used in tests for medicines, which have to be repeated on other species”.

The substances studied act on the hypothalamus, a region of the brain that controls the production of two hormones that help to determine sex and regulate the functioning of the ovaries and of the testicles – the luteinizing hormone (LH) and the follicle stimulating hormone (FSH). In mice, the hypothalamus matures between the 18th day of gestation, which lasts 22 days, and the first week after birth. It is a critical period, corresponding to the last three months in a woman’s pregnancy and the beginning of the breast feeding of the baby.

It is when the so-called masculinization of the thalamus takes place, which is them getting ready to take on, years later, in puberty, the masculine pattern of producing LH and FSH. Independently from the sex determined at the fecundation by the X and Y chromosomes, the hypothalamus of the fetus has feminine characteristics. In the masculine sex, this pattern is altered only at the end of pregnancy by this region of the brain being sensitized, which is set off by the masculine hormone, testosterone.

Antiallergics
When Maria Martha decided to research the effects of antiallergics on the offspring of mice, in the 80’s, there were few studies into drugs that act on histamine, a substance that unleashes inflammation, but, in the central nervous system, acts as a neurotransmitter and regulates the release of hormones. The initial focus was diphenhydramine, an antiallergic that is transformed dimenhydrinate, one of the active ingredients of Dramin, used by pregnant women to fight sickness. The tests carried out at USP showed that this substance, applied in pregnant mice, may retard puberty.

To discover whether diphenhydrine interfered with the sexual behavior of the offspring, Maria Martha worked with Silvana Chiavegatto, of USP’s Institute of Biomedical Sciences, on experiments in which pregnant mice received, during gestation, daily doses four times higher than those indicated for humans. The result: when they reached adulthood, the males showed reduced sexual behavior: they would try many times to copulate with females, and took more time to ejaculate. Later studies showed that the effect of these substances was long-lasting and affected sexual differentiation in the offspring, demasculinizing them. In the tests where the rodents are put one by one into an arena, the males, generally less interested in the environment for not being responsible for feeding the offspring, started to explore it in similar way to the females. Maria Martha and Silvana proved that diphenhydrine altered the system of the neurotransmitter dopamine, associated with controlling the masculine sexual behavior.

The advance of antihistamines led Maria Martha to assess astemizole, then used to treat rhinitis and conjunctivitis. Applied to mice during gestation or in the first week of breast feeding, in doses ten times higher than those indicated for humans, astemizole acted in a similar way to diphenhydramine on the sexual behavior of the male offspring. Regardless of the period when it was applied, it altered the way dopamine worked and affected the nervous system of the fetus, according to an article published in the March/April issue of Neurotoxicology and Teratology.

Chemical stress
In parallel, another group of veterinary scholars from USP, coordinated by Yara Almeida, worked with phenvalerate, an insecticide from the type II class of pyrethroids – substances that are toxic for the central nervous system and that account for 30% of the world production of pesticides. Yara found that phenvalerate, and the deltametrine pyrethroid as well, interferes with the activity of the Gaba neurotransmitter, which is associated with learning and sexual behavior, causing a sort of chemical stress set off by the release of corticosterone, the stress hormone. In the 70’s, American researchers proved that stress generated by environmental factors, such as the cold and the lack of food, caused demasculinization and feminization in mice.

It was now shown that chemical stress acted in a similar way. In the experiments, the pregnant mice were given doses of phenvalerate 500 times higher than the acceptable daily dose for humans, in the 18th day of gestation and on the first five days of breast feeding. Afterwards, the male mice, set to mate with females, took longer to ejaculate (they would cover the female mice 22 times before having an ejaculation, against 15 times for the males whose mothers had not been given the pesticide), and they showed some 40% less testosterone than the control group.

To study chemical stress, Maria Martha and another researcher from the school, Maria Rita Pereira da Silva, created an experimental model with picrotoxin, a toxic substance extracted from a climbing plant called anamirta (Anamirta cocculus), which inhibits the activity of the Gaba, in a similar way to pyrethroids. Its effects soon became evident. When put together with females, the mice exposed to picrotoxin in the perinatal period (in the uterus and right after birth) took twice as long to attempt their first mating, and three times more to achieve their first copulation than the offspring that had no contact with the compound. The mice from the first group would mate with the females more often before achieving their first ejaculation, showed half the normal level of testosterone and weighed less than normal males. With the female offspring, there was masculinization: the little female mice whose mothers had contact with picrotoxin did not menstruate and were permanently on heat.

According to Maria Martha, picrotoxin and the type II pyrethroids interfere in the workings of the Gaba system by two mechanisms: one associated with the mother, and the other with the offspring. In the latter, they inhibit the action of the neurotransmitter that controls the production of the masculine sexual hormone by the testicles – essential for masculinizing the hypothalamus at the end of gestation and in the first few hours of life. In the pregnant mice, the alteration in the Gaba system reduces the quantity of the gonadotropin-releasing hormone (GnRH – which stimulates the production of testosterone by the testicles) available in the colostrum, the liquid produced by the mammary glands in the first few days after the birth. Receiving less GnRH, the offspring can produce testosterone before or after the critical moment of the masculinization of the hypothalamus, or produce less potent masculine hormones.

The effects of lead
Another toxic substances that acts in a similar way is lead. In a study in which this substance was given to mice during the first week of lactation, Maria Martha and Marcela Gonçalves Sant’Ana (who is studying for her doctorate) saw that this metal affected the sexual behavior of the male offspring, by inhibiting the action of the GnRH. The administration of this substance in a concentration of 0.1% brought about the demasculinization of the offspring, while the offspring of mice that consumed lead at t level of 1% showed another undesirable effect, premature ejaculation.

The picrotoxin model proved extremely useful to understand the masculinization of the brain: it has now indicated that the most important moment in this process, at least in mice, is the first few hours after birth. “The model makes it possible to study not only the alterations in behavior, but also in which areas of the hypothalamus the modifications are taking place”, comments Oduvaldo Marques Pereira, from the Institute of Biosciences of the São Paulo State University (Unesp), in Botucatu, which supplemented the USP model.

Pereira recently demonstrated the feminization of mice exposed to chemical stress in the perinatal period. Although in the human species the determination of sexual behavior is more complex, as there as social and cultural influences, Pereira explains: On the basis of experiments with mice, I believe that human beings are also subject to alterations in behavior caused by exposure, occupational or deliberate, or by the consumption, chronic or occasional, of medicines in the perinatal period”.

Herbicide produces hermaphrodite toads

USP’s work on the impact of pesticides on the sexual development of mice fits in with a line of research that has recently created another worrying piece of news. An American study, published in April, showed that exposing the larvae of male toads to doses 30 times less than the legally permitted dose of a kind of herbicide, atrazine, encourages hermaphroditism in these animals when developing. Due to the contact with the chemical product, used in plantations to kill weeds, tadpoles of the Xenopus laevis species showed characteristics of both sexes.

“The males had ovaries in their testicles and their vocal organs are much smaller than usual”, explains Tyrone Hayes, of the University of California in Berkeley, the main author of the work, published in the Proceedings of the National Academy of Sciences. “Their reproductive system is not normal”.

Atrazine, the best selling herbicide in the United States in its category, also affects adult male toads: it decreases tenfold the level of testosterone, the principal masculine sexual hormone. The toads become so effeminate that they have less testosterone than their partners of the opposite sex.

For Hayes, sexual disorders caused by tiny doses of atrazine, in the order of 0.1 ppb (parts per billion), far lower than those found in rain or on the plantations, may be one of the causes of the world-wide decline of amphibians. Atrazine has been used for 40 years on crops like corn and soya in 80 countries, Brazil included. “There doesn’t seem to be any environment free from atrazine”, says Hayes. “This product is so disseminated that it endangers aquatic systems.”

The Project
Assessment of the Influence of Environmental and Chemical Factors on Parameters Connected with the Reproduction of Mice (nº 96/04273-4); Modality Thematic project; Coordinator Maria Martha Bernardi – College of Veterinary Medicine at USP; Investment R$ 167,193.39

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