Assault on autism: scientists target drugs and other environmental agents that may play a role.

Beth Crowell remembers the day in 1989 when her triplets, Casey, Andrew, and Erin, were about 15 months old. Crowell put Erin down on the floor to crawl. "But she just sat there, fixated on the red shag carpeting," says the Housatonic, Mass., mother of four. The toddlers were often sick, and "none of them made eye contact," Crowell recalls. A medical evaluation was devastating: All three babies had autism. Children with autism typically have trouble communicating, interacting socially, and controlling their behavior. Those most severely affected seem to live in a world of their own. Various treatments sometimes reduce symptoms, especially if children are diagnosed early. But there is no cure for autism, which has baffled the medical community since the disorder was first described in 1943.

The Centers for Disease Control and Prevention (CDC) recently estimated that 1 of every 1,000 children may have autism, or 1 in 500 if those with autism-related disorders such as Asperger syndrome are included.

For years, Crowell combed the medical literature trying to figure out what might have gone wrong in her triplets. She doubted that a genetic mutation was solely responsible. Crowell came to suspect that terbutaline, a drug she had taken during pregnancy to prevent premature labor, might have played a role.

A team of researchers in Baltimore found her assertion plausible. They knew of experiments showing that rats exposed to terbutaline before birth had brain abnormalities. More recently, they completed a yet-unpublished clinical study that found a higher-than-expected incidence of autism in both children in sets of fraternal twins whose mothers also took terbutaline during pregnancy. The investigators are Andrew Zimmerman of the Kennedy Krieger Institute, independent researcher Susan Connors, and researchers at Johns Hopkins Medical Institutions.

This research reflects a shift in scientific thinking about what causes autism, and a push to look harder at potential environmental influences.

"For years, the emphasis [in autism research] has been on neurobiology and genetics," says Michael Cuccaro of Duke University in Durham, N.C., a psychiatrist specializing in autism. "It was thought we could identify the causes if we could understand those connections, but we're still left searching for causes. There was a missing piece of the puzzle, which was the environment."

Some scientists are convinced that environmental factors must be at play because autism appears to be increasing rapidly. They argue that genetic factors alone can't account for such rapid growth. For example, California Department of Developmental Services data show that autism cases in the state more than doubled between 1987 and 1998. Scientists from the CDC found a 10-fold increase in autism in Atlanta from 1986 to 1996.

However, perinatal epidemiologist Lisa Croen of Kaiser Permanente's Division of Research in Oakland, Calif., says that some of the apparent increase probably reflects inconsistencies in data-reporting methods and changes in diagnostic criteria over the past 15 years.

A highly controversial piece of the autism picture in the past decade has been the issue of whether childhood vaccines can trigger the disorder. Suspicions arose because autism is often diagnosed around the time when children receive a series of routine vaccinations. A mercury-based vaccine preservative called thimerosal seemed a likely culprit.

In the past 5 years, thimerosal has been phased out of most pediatric vaccines, and a committee of the National Academies' Institute of Medicine in Washington, D.C., has consistently found no conclusive evidence for an autism-vaccine connection. But two new studies are reviving the argument that thimerosal can act as an environmental factor promoting autism in certain children

Geneticist Thomas Wassink of the University of Iowa in Iowa City says that most researchers studying the genetics of autism now assume that the disorder is caused by interplay between genes and factors from outside the body. He speculates that environmental factors trigger the disorder in children in whom 5 to 15 genes have created an underlying susceptibility. Gene hunters are homing in on several autism-related genes, he says.

Much of the current research on autism is in early stages. Payoffs in treatments or preventive measures are likely to be years to decades away.

MATERNAL MEDIATION Environmental agents under scrutiny in autism research include drugs, vaccines, viruses, and poisonous substances such as lead and mercury. "There certainly isn't a shortage of environmental suspects that may play a role in autism," notes Andy Shih, chief science officer of the National Alliance for Autism Research in Princeton, N.J. "But these are not all necessarily artificial or manmade and may have to do with influences in the womb."

Zimmerman, a pediatric neurologist, is one of many specialists who think that environmental influences in utero may contribute to autism by disrupting normal early development. To a fetus, any effect from outside the womb--hormones triggered by a mother's stress, for example--is environmental. "It's anything that affects pregnancy," says Zimmerman.

Isaac Pessah, director of the Center for Children's Environmental Health and Disease Prevention Research at the University of California, Davis, agrees. He also points out that newborns and infants are especially vulnerable to the damaging effects of toxic exposures because the human nervous and immune systems "undergo considerable remodeling in the first 2 years of life"

Some scientists suspect that maternal viral infections are one of the principal noninherited causes of autism. Epidemiological studies have found a significantly increased risk of autism in the offspring of mothers exposed to the rubella, or German measles, virus early in pregnancy.

In the Jan. 1, 2003, Journal of Neuroscience, scientists led by Paul H. Patterson of the California Institute of Technology in Pasadena reported that when pregnant mice were infected with a modified human-flu virus, they produced offspring that, as adults, behaved in ways similar to those of many autistic children. Compared with a control group, the affected mice interacted less and were unusually anxious under mildly stressful situations and around unfamiliar objects.

The scientists also found unusually low numbers of critical signaling components, called Purkinje cells, in brain tissue of the affected mice. Autopsies of people with autism have revealed fewer than normal of these cells.

In an upcoming International Journal of Developmental Neuroscience, Patterson's group reports that altered brain development in the mice doesn't appear to occur as a direct result of viral infection in the fetus. Instead, "there's evidence it's related to a natural immune response in the mother, [but the] mechanism is something we're still working on,' says Patterson.

Some of the molecules that the mother uses to fight the virus may be crossing the placenta and affecting brain development in the fetus, he explains.

If so, the problem wouldn't be specific to the flu virus. "Lots of kinds of infection could lead to the same effects," Patterson says.

IMMUNITY ROLE? Support is growing for the idea that immune system problems in a pregnant woman or developing child set the stage for autism.

Zimmerman and his colleagues recently found that rheumatoid arthritis and other autoimmune disorders, in which the immune system attacks parts of the body, are unexpectedly common in families of autistic children. Zimmerman says that other studies have shown that from 30 to 70 percent of antistic children have subtle immune system abnormalities, although such children aren't exceptionally vulnerable to everyday infections.

To study whether impaired immunity might put some children at risk of developing autism after being exposed to thimerosal, Mady Hornig of Columbia University and her colleagues did experiments using mice vulnerable to autoimmune diseases. The researchers injected newborns of this susceptible strain and of two other strains with thimerosal alone, with a thimerosal-vaccine combination, or saline solution. The doses were comparable to those that children receive in typical vaccinations.

In the September Molecular Psychiatry, Hornig's team reports that virtually all the mice in the immune-compromised strain that received either form of thimerosal showed autismlike symptoms. They behaved oddly and had delayed growth and brain abnormalities. The other two strains of mice showed no such effects.

Some scientists caution against reading too much into the findings. Epidemiologist Craig Newschaffer of Johns Hopkins says that animal experiments such as this are important to determine the physiological effects of exposure to toxic substances. But, he notes, it's impossible to say with certainty that lab animals exhibiting certain kinds of behavior have autism or that what happens in lab animals translates to people. "We have to keep in mind that these are largely preliminary studies," he says.

POTENTIAL PATHWAYS As in other diseases, finding the roots of autism is challenging because things can go awry at so many points on the long and complicated road to normal human development.

In the April Molecular Psychiatry, neuropharmacologist Richard Deth of Northeastern University in Boston and his colleagues described a biochemical pathway that they say is an avenue by which thimerosal and other compounds could cause neurodevelopmental disorders such as autism.

When the researchers exposed human neuronal cells to low doses of thimerosal, the chemical activity called methylation dropped significantly. Deth explains that methylation, in which single carbon atoms are transferred from one molecule to another, plays a central role in normal patterns of gene expression. Therefore, he says, "it is no wonder that [agents] capable of interfering with DNA methylation could cause developmental disorders such as autism."

On Oct. 5, Deth described his findings to a House of Representatives Labor and Health and Human Services subcommittee in Washington, D.C. He told the panel that his team recently determined the specific molecular mechanism by which thimerosal inhibits methylation. An enzyme critical to methylation, methionine synthase, uses an active form of vitamin [B.sub.12] to complete its chemical function, Deth explained. Thimerosal interferes with the conversion of dietary forms of [B.sub.12] into the active form and so impedes DNA methylation and disrupts some normal gene actions.

The work, Deth says, suggests that children with mutations in genes that encode methylation-related enzymes are at higher risk of damage from toxins.

The researchers found that exposing neuronal cells to lead, ethanol, and aluminum also disrupts methylation, but through different mechanisms.

Meanwhile, Connors, Crowell, and their Baltimore colleagues hypothesize that the drug terbutaline, which is usually used for asthma, leads to autism by interfering with beta 2 adrenergic receptors, cell-membrane proteins that play a major role in brain development. Animal studies of terbutaline have shown that it overstimulates beta 2 receptors, says Connors.

As a result, she explains, the receptors produce excess cell-to-cell signals, confusing the development process. Connors and her colleagues suggest that this effect alters nervous system growth, putting some children at risk of developing autism when exposed to various environmental insults.

"We don't want to emphasize terbutaline exposure per se [because] there isn't only one mechanism that can overstimulate beta 2 receptors," says Connors. She notes that stress hormones, for example, bind to beta 2 receptors, which suggests that unusually high maternal stress during certain stages of pregnancy might also contribute to some cases of autism.

Crowell, whose triplets are now teenagers, has been a coinvestigator of the Baltimore studies.

She says that life as a parent of autistic children "continues to be challenging. Our children can learn, so we're constantly having to advocate for education. Being vicious in advocating for our children pretty much consumes my day."