Part III: Here, There, Everywhere: Chasing the plastic impostors
Introduction | Omens
Working with cells in tissue culture can be a tricky business. There is only one way of doing things--impeccably. Any lapse in discipline--the least hint of slop-piness--can ruin weeks, months, even years of work. To eliminate potential problems, Sonnenschein and Soto took elaborate precautions and never had a problem-until that final week of 1987.
Sonnenschein had prepared a series of multiwelled plastic plates, placing breast cancer cells in twelve small cups and then adding varying levels of estrogen or of the estrogen-free serum to the tiny cell colonies. Four days later, the two scientists returned to the lab to see how the cells had fared. According to the routine, they would examine the cells under the microscope before transferring them from the plates to special counting vials for tallying by an electronic particle counter. Over the years, they had done hundreds of variations of this experiment.
Somehow the first plate didn't look right, so Sonnenschein adjusted the microscope and looked again. The whole plate--every single colony growing in the specially modified blood serum--was as crowded as a subway train at rush hour. The estrogen-sensitive breast cancer cells were not only multiplying, but they were doing so regardless of whether estrogen had been added. In all their years of cell work, Soto and Sonnenschein had never seen anything like it. Something had gone seriously wrong. It had to be some sort of estrogen contamination, because non-estrogen-sensitive cells in other experiments were behaving as expected.
Carefully preparing another batch of plates with breast cancer cells, the two scientists again saw galloping proliferation. It wasn't a fleeting event. The mysterious source of contamination had to be somewhere in the lab. To trace it would mean a tedious process of elimination. It would be four long, frustrating months of checking and rechecking procedures and equipment before the two finally tracked down a source--and two whole years before they were able to put a name to the chemical contaminant that was causing the cells to proliferate. The source was the orange-capped Corning centrifuge tubes that the researchers had always regarded as benign and inert. But something in the tubes appeared to be biologically active.
These findings prompted Sonnenschein and Soto and other Tufts representatives to meet with Corning officials on July 12, 1988. At that meeting, Soto and Sonnenschein learned that Corning had recently modified the composition of the plastic resin in the tubes to make them less brittle. The company had not changed the catalog number on the item, however. When Soto asked about the chemical content of the new resin, Corning declined to disclose the information on the grounds that it was a "trade secret".
Worried about the possible broader implications of their discovery, Sonnenschein and Soto determined to purify the offending compound and to make a preliminary identification using mass spectrometry analysis. Finally, at the end of 1989 they had a definitive answer: the active component in the plastic was p-nonylphenol, part of a family of synthetic chemicals known as alkylphenols and commonly added to polystyrene and polyvinyl chlorides (PVCs) to make them more stable and less breakable. The plastic centrifuge tubes in which Soto and Sonnenschein had stored serum had been of polystyrene--a plastic that, depending on the manufacturer, may or may not contain nonylphenol.
Searching the scientific literature, the investigators found bits and pieces of information that only heightened their concern. One study reported that PVCs containing alkylphenols were used in the food-processing and food-packaging industries. Another investigation found nonylphenol contamination in water that had passed through PVC tubing. Soto and Sonnenschein even discovered that nonylphenol had been used in synthesizing nonoxynyl-9, a compound in contraceptive cream. Yet another experiment demonstrated that nonoxynyl-9 broke down into nonylphenol in laboratory rats.
To ascertain whether p-nonylphenol acted like an estrogen in living animals--not just in a lab dish--Soto and Sonnenschein injected the substance into rats. They found that in female rats without ovaries, p-nonylphenol caused the lining of the uterus to proliferate (as would have happened had the animals been given natural estrogen).
The researchers also learned that alkylphenol polyethoxylates (chemicals found in many detergents, pesticides, and personal-care products) can break down into nonylphenol and other chemicals that mimic estrogens when they encounter bacteria in animals' bodies, in the environment, or in sewage treatment plants. Although alkylphenol polyethoxylates have been in wide use since the 1940s, they came under scrutiny in the 1980s because of their toxic effect on aquatic life. By the late 1980s, several European countries had already banned their use. In 1990, the United States was still using more than 450 million pounds annually.
When Soto and Sonnenschein published their findings in 1991, even veteran investigators of hormone-disrupting chemicals were shaken. For years, the ongoing discussion about possible human health risks from synthetic chemicals had been based on the assumption that most human exposure comes from chemical residues, primarily pesticides, in food and water. Now Soto and Sonnenschein had discovered hormone disrupters where you would least expect them--in ubiquitous products made from materials considered benign and inert. Here was glaring evidence of our vast ignorance about the dangers in our everyday environment.
While Soto and Sonnenschein were chasing contamination in their lab, a similar drama was unfolding at the opposite end of the country, at Stanford University School of Medicine in Palo Alto, California. In this case, too, an estrogen-mimicking compound was traced to plastic lab equipment--but the culprit substance had not been linked with polystyrene products or nonylphenol. The Stanford team found another estrogen mimic, bisphenol-A, leaching from polycarbonate, an entirely different kind of plastic. Polycarbonate is used in the manufacture of lab flasks, as well as many consumer products, such as the giant jugs used to bottle drinking water.
Here again, the discovery was accidental and occurred only because the scientists were conducting research with estrogen-sensitive cells--in this case, yeast. In the course of their experiments, the Stanford researchers, headed by endocrinologist David Feldman, found a contaminant binding to a yeast protein. The estrogen mimic proved to be the bisphenol-A in the lab flasks they used to sterilize water.
In a 1993 paper, the Stanford team reported their discovery amid their discussions with the manufacturer of the polycarbonate, GE Plastics Company. Apparently aware that bisphenol-A leaches out, particularly if exposed to high temperatures and caustic cleaners, the company had developed a special washing regimen to eliminate the problem. Stanford began working with GE and soon discovered that the company's chemical assay could not detect bisphenol-A at levels below ten parts per billion. Yet two to five parts per billion of bisphenol-A was enough to prompt an estrogenic response in cells in the laboratory. GE officials contended, however, that polycarbonate containers are unlikely to leach bisphenol-A in normal use because they would not be subject to the high temperatures required for sterilization.
Spurred by the Tufts researchers' report of biologically active plastics, scientists at Spain's University of Granada decided to investigate the plastic coatings that manufacturers use to line metal cans. These often inconspicuous coatings are because of concerns that met-als might contaminate the food or impart a metallic taste. Such plastic linings are reportedly found in 85 percent of food cans in the United States and about 40 percent of those sold in Spain. The brother-and-sister team of Maria-Fátima Olea, a food toxicologist, and Nicolás Olea, a physician specializing in endocrine cancers, analyzed twenty brands of canned foods purchased in the two countries. They discovered bisphenol-A (the same chemical that Stanford researchers had found leaching from polycarbonate lab flasks) in stunningly high concentrations in such canned foods as corn, artichokes, and peas. Bisphenol-A contamination was detected in about half the canned foods they analyzed. In some instances, the cans contained as much as eighty parts per billion--twenty-seven times the amount that the Stanford team reported was enough to make breast cancer cells proliferate. Even though synthetic estrogens are less active than natural hormones, at such levels they may be contributing significantly to human exposures. Biologically active plastics were leaching from "metal" cans, where one would not expect to find plastic at all.
Colborn, T., D. Dumanoski, J. P. Myers. 1996. Hormonal Sabotage. This article and the ones linked to it was originally published in Natural History, March 1996, 105(3):42-49. Excerpted from the book Our Stolen Future.
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