Molecular mechanisms that underlie birth defects

Although only a small percentage of environmental agents has been shown to cause birth defects in humans, all new drugs and a limited number of chemicals are tested in animals to assess teratogenic risk. These studies are expensive, and the large number of agents that require testing creates a demand for less expensive methods.

Less expensive tests have been proposed, but they are unlikely to be useful predictors of developmental toxicity until science has a better understanding of how teratogens actually interfere with the process of prenatal development, argues CHDD research affiliate Dr. Philip Mirkes. "We don't know enough about the biology of normal development to really know the appropriate questions to be asking of these tests," he says. Most human teratogens have been identified based on population studies that look back in time and correlate prenatal exposures to particular environmental agents with patterns of congenital malformations. On the basis of data from the population prenatally exposed to thalidomide, we know it is a potent human teratogen. Yet, Mirkes notes, poor understanding of mechanisms of teratogenesis means we still don't know why.

For the past 18 years, Mirkes, research professor of pediatrics, has been investigating fundamental cellular processes with the goal of uncovering how the basic mechanisms of development are disrupted by exposure to developmentally toxic environmental agents. Answers to the questions he is asking will provide a foundation for improved methods for screening potentially teratogenic agents. They will also pave the way for strategies that could protect against effects of harmful exposures.

"Even though what I do is very basic and at times seems very esoteric, I think there is a real possibility that it could strengthen our ability to prevent birth defects. That's the goal--what motivates us to do what we do," says Mirkes.

Cell death, one of the processes Mirkes is investigating, is an important part of normal development. Cell death is associated with the development of most tissues and organs, he explains. "For example, we don't have webs between our fingers because all those cells die away during development and we end up with digits."

A limited amount of cell death is critical to normal development, but the process of teratogenesis involves cell death beyond what is typical. Researchers have found evidence of large amounts of cell death in animals exposed to agents known to cause various tissues to develop abnormally.

"The process of cell death is actually controlled by very specific molecules and genes," says Mirkes. "Over the past couple of years we've been looking at some of these key genes--how they regulate cell death in the embryo and how they might be affected by agents that cause birth defects."

Currently Mirkes is studying a gene called Bcl-2 and the protein it synthesizes, known to be a key factor in controlling whether a cell lives or dies. To learn more about the specific role of this gene in response to teratogenic agents Mirkes' lab is beginning work with a transgenic mouse model, in which the Bcl-2 gene has been "knocked out," so it is not expressed--the protein that blocks cell death is not produced. With this model, they can test the hypothesis that an embryo without this gene and its products may be much more susceptible to a particular exposure than an embryo in which the gene is fully functioning.

Another major focus of Mirkes' research is to elucidate the mechanisms that an embryo can call upon to protect itself from agents that can cause cell death and, subsequently, abnormal development. For the past several years, he has been investigating a group of cellular proteins known as heat-shock proteins. Mirkes has found that these proteins can protect an embryo from the stress related to the very thing that induces their production--elevated maternal body temperature, also called hyperthermia, caused by high fever. In humans, hyperthermia that occurs during embryonic brain development has been linked with malformations of the central nervous system.

Mirkes and his colleagues have shown that while acute exposures to elevated temperatures result in abnormal development, less severe exposures can actually protect embryos from a subsequent acute exposure. It seems that production of a certain amount of heat-shock proteins creates thermotolerance. Other research suggests that heat-shock proteins may also protect against the effects of other teratogenic agents.

Mirkes is seeking to learn more about how a particular heat-shock protein provides protection by studying it directly in a transgenic mouse model. His investigation is focusing on Hsp-70, the gene whose protein has been most clearly implicated in the protection process. Hsp-70 is normally silent. It only "turns on" and begins protein synthesis in response to stress, such as exposure to elevated temperature or other specific teratogenic agents. By studying an animal model in which Hsp-70 is turned on all the time, Mirkes hopes to answer basic questions that may someday lead to therapeutic interventions for use after a teratogenic exposure. "If, for example, a woman has a very high fever, we might be able to give her something that very quickly stimulates production of Hsp-70 in her embryo," suggests Mirkes. In addition to his own research, Mirkes takes an active role in fostering communication among researchers, clinicians and policy-makers. He is current president of the Teratology Society, a national organization composed of members from four professional areas: clinicians, mostly pediatricians and obstetricians; basic scientists; individuals who work in the pharmaceutical and chemical industries, and members of government agencies. The organization publishes a journal, shares scientific findings at its annual meeting, and, with assembled expertise from its various areas of membership, prepares position papers on issues related to teratogens.

Mirkes also directs a training program at the UW for clinicians. The program trains pediatricians who have completed their residencies in the basic science needed to conduct investigative research related to birth defects.

Understanding what is occurring at the molecular level is important, according to Mirkes, but he acknowledges that it is not the only way to prevent birth defects. He points to growing use of folic acid supplements.

Epidemiological studies showed that women who have had one baby with a neural tube defect dramatically reduced their chance of having a second baby with this defect if they took folic acid prior to and during the early part of gestation. Folic acid is now widely recommended as a supplement during pregnancy and even cereal boxes tout that they contain folic acid, Mirkes points out.

"Although we don't really know the basic science of what folic acid is doing to protect that embryo from spina bifida, I think this finding will have a huge impact," says Mirkes. "In the end, however, to really understand why these things work or don't work, we need to understand the basic science."

These dark-field images from one of Mirkes' studies depict a tissue section through a rat embryo limb bud at day 15. The section has been stained using the TUNEL method, which detects DNA fragmentation associated with a form of cell death called apoptosis. The images reveal that apoptosis is associated with this stage of normal limb development. Numbers refer to the individual digits of the developing limb. The white arrows indicate apoptosis occurring in the tissue between the digits. The black arrows point to apoptosis at the interphalangeal spaces, which give rise to joints. The image at the lower right is a greater magnification of the area between digits 2 and 3.