Leaching plastics throw lab assays

Two compounds ubiquitously present in disposable linkurl:lab plastics;http://www.the-scientist.com/2008/11/1/23/1/ -- from test tubes and pipette tips to 96-well plates-- may be wreaking havoc on biomedical experiments, a study in Science reports this week. The study provides "clear evidence of these compounds leaching out of plastics," said linkurl:Andrew Holt,;http://www.pmcol.ualberta.ca/personnel/faculty/AHolt.htm a pharmacologist at the University of Alberta, Canada and main author. The compounds affect the activity of receptors, enzymes and ion channels -- "three main types of molecules responsible for controlling virtually everything in the body." Holt identified the two troublesome compounds linkurl:the hard way;http://www.the-scientist.com/community/posts/list/18.page -- when they messed up his own experiments. His group studies linkurl:monoamine oxidase;http://www.the-scientist.com/article/display/19648/ (MAO) B, an enzyme that regulates the neurotransmitter dopamine and is a potential target for Parkinson's disease treatment. The group was testing a drug inhibitor of MAO-B, but were getting inconsistent results. Often, they'd see strong inhibition of MAO-B at drug concentrations they knew were too low to be causing such potent effects. Initially, Holt suspected contamination in some step of the assay "After several months of tearing our hair out," he said, he and his lab technician finally traced their irregular results to the plastic tubes in which they mixed reagents. It seemed that reagents mixed in some plastic tubes produced strange results, while those mixed in others did not, leading Holt to suspect that some compound was leaching from the plastic. Indeed, when they washed drug-free control buffer through the brand of plastic tubes they usually used and ran it through their assays, the solution strongly blocked MAO-B activity. They began to test other plastics in the lab, and found that some, but not all, showed a similar effect, but which ones were problematic was hard to predict. For instance, the 1.5 ml microfuge tubes the group used that were made in Germany did not affect their experiments, but smaller tubes, sold by the same company but manufactured in Italy, did. The problem wasn't limited to tubes, either - pipette tips also caused the effect. Using mass spectrometry, Holt's group identified the problematic compounds leaching from the plastics as quaternary ammonium biocides and a fatty acid amide, oleamide. The researchers then obtained the compounds themselves, to test directly in their assays, and found they were potent MAO-B inhibitors. Biocides are synthetic chemicals that can act as detergents, and oleamide occurs naturally in blood and brain tissue; both types of compounds are used in plastic manufacturing. "It turned out these compounds are deliberately included by manufacturers to alter properties of molten plastics," said Holt. As a result, "their presence [in lab supplies] is by and large unavoidable." Suppliers likely use different versions of the compounds as processing agents, and different types of the compounds would probably affect different assays differently, explaining why some batches of plastic products affected his assays while others did not. Oleamides are endogenous chemicals that have been found to bind GABA receptors, canabinoid receptors, and several subtypes of serotonin receptors, so Holt examined experiments of a colleague in his department, Susan Dunn, who studies the effect of drugs on GABA receptors. They conducted similar tests of the plastics she used in her assays. "Sure enough we saw very substantial inhibitory effects on ligand-binding on GABA receptors," he said. Nothing in the literature predicted oleamide's effect on MAO-B, he noted, "so I'd say there's a reasonable chance that there may well be other [affected assays] that we couldn't even predict." In experiments with his group's and her group's assays, the effect varied between batches of plastics, and, to a lesser degree, even between individual test tubes. Adding to the unpredictability, he said, "the tubes that we found had greatest effect on our enzyme had no effect on Dr Dunn's binding assay," and vice versa. Holt began checking with others in his department, which contains some 20 labs, and found that he and Dunn weren't the only ones encountering the problem -- a third lab also suspects the chemicals are having an effect. Can those numbers - 3 out of 20 - be extrapolated to biomedical researchers in labs worldwide? That's unclear, Holt said, but "if the numbers did pan out to be anything like that, you're looking at very very large numbers of labs who are going to be affected." The solution to the problem will have to come from plastics manufacturers, he said. "If they are aware that this is now a problem, without really changing too much what they do and how," they should be able to make several different lines of lab plastics using specific oleamides and biocides. Researchers would then be able to test their set-ups and make sure they're using a variant of the chemicals that doesn't affect their assay. But until then, he said, "each individual researcher really is going to have to make themselves aware of [these chemicals] and screen them in their own particular assay systems." His own group tests each batch of plasticware and thoroughly washes plastics just before use in an experiment. That's no good as a long-term solution, however, since it takes about two hours to wash out the plastic before a three hour experiment, almost doubling the time the experiment takes, he said. "It's a mess."Editor's note (November 7): A previous version of this article erroneously mentioned 92-well plates when meaning 96-well plates. The Scientist regrets the error.

Leaching plastics throw lab assays

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