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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 c

By | November 6, 2008

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.
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Comments

Avatar of: anonymous poster

anonymous poster

Posts: 2

November 7, 2008

Is there any effect in using glass instead (other than a potential increase in disposables price)?
Avatar of: JOHN SPEVACEK

JOHN SPEVACEK

Posts: 1

November 7, 2008

Leaching is only half the problem with plastic. How about chemicals/biologics/whatever-you-are-really-interested-in sticking to the plastic? See for example: "Binding of taxol to plastic and glass containers and protein under in vitro conditions" Journal of Pharmaceutical Sciences (85)1996 29-31\n\n
Avatar of: m woodman

m woodman

Posts: 2

November 7, 2008

bio labs seem to have widely gone to plastic over the years. Glass shouldn't leach anything significant, but adsorption is invariably a concern. It has been addressed in certain glass products like autosampler vials but I'm not sure about test tubes and other disposables. \nIt is hard to imagine glass tubes in a high force centrifuge, and the potential of injuries complicated by bio-exposure no doubt play a role in avoiding glass.\nI think the answer lies in communication with the supplier and the purchasing department. Price drives many of the decisions and this seems like a situation that demands a careful look at product quality before starting a study.
Avatar of: anonymous poster

anonymous poster

Posts: 1

November 7, 2008

The person who actually made this discovery deserves to be recognized.

November 7, 2008

I do not know if just by sterilization of plastic ware could "clean up" those problematic chemicals. Maybe chemists have the answer.
Avatar of: anonymous poster

anonymous poster

Posts: 107

November 8, 2008

What about all of us out here eating and drinking out of plastic containers?
Avatar of: tian xia

tian xia

Posts: 34

November 9, 2008

After culturing cell lines for a while,the cells look different. One of my senior co-worker always say, they become old, get a new vial from liquid nitrogen. Most of time I blame myself for not being "caring" enough. Although I still think I am the deciding factor, according to this article, I am actually adding a treatment to the cells, over time that could make a difference.
Avatar of: Christopher Lee

Christopher Lee

Posts: 50

November 9, 2008

We mentioned a similar problem with ultrafiltration membranes in the article 'Pitfalls in demonstrating an endogenous ligand of imipramine recognition sites', C. R. Lee et al Biochem Pharmacol 1987, 36(6), 945-949.\n\nThe culprit was tentatively identified by gas chromatography as tributylamine, but this could have been a pyrolysis product of a quaternary amine.\n\nAll you can do is be aware of such problems.\n\nAs a chromatographer, I endorse the comments about adsorption to plastic and glass, and add that some batches of glass vials straight from the glass-blowing mould can be worse than others, and very alkaline.\n\nRegards
Avatar of: anonymous poster

anonymous poster

Posts: 2

November 9, 2008

All the more reason to ban plastics from food serving items. \n\nRecently the CBC aired a documentry on reduced male birth rates caused by products of the chemical industry in a certain part of Canada. With corn based "plastics" becoming cheaply available, why are we still using petroleum based plastics in tissue culture and food service industry? All that foreign oil is slowly poisoning us.\n\nBiodegradable corn based plastics also reduce landfill issues and roadside litter from food cartons. Time for us to have a spine and do whats right.
Avatar of: R CALDWELL

R CALDWELL

Posts: 6

November 10, 2008

Working in a molecular lab, we have always known about problems with labware, reagents and suppliers. The biggest problem is that no one talks about it or worse tries to push the blame onto user error. We work with a cell line that is normally robust, but recently had numerous problems. We finally tracked the culprit down to the media we purchased. While we bought from the same company, that company relocated its plant to another production facility in another country (cost savings). Starting with the very first batch from this new facility, the media no longer supported robust healthy growth. It took months for the company to clue us in on this new variable yet they still tried to claim user error even after we gave them the data proof positive. We have since revised our media screening method to be more sensitive to subtle compositional changes in wake of this incident and have changed media suppliers. \nThe problem is, we can't do that for every reagent and every piece of labware we use. The time and costs would be staggering. We simply know in advance that on a 96-well PCR plate for example that there will be "interference" beyond our realistic controls.
Avatar of: anonymous poster

anonymous poster

Posts: 1

November 10, 2008

I'm not at all surprised by these findings, and to make matters worse, glass has problems too.\nIn graduate school we did a lot of work with bacterial luciferase reporter assays. The experiments were conducted in 250mL pyrex flasks and the inducer for gene expression was salicylate. People in the lab were getting a lot of background and traced it to the salicylate adsorbing to the flasks and contaminating later experiments. This occurred even after washing several times. We finally opted to bake all of the glassware in a muffle furnace prior to use, which finally resolved the issue. \n\nAdditionally, most of the enzymes currently used(particularly DNA binding enzymes) are contaminated with DNA from the host. Not a big deal usually, but if you're trying to amplify environmental samples with universal 16S primers for example, it can lead to problems.
Avatar of: JAY THAKAR

JAY THAKAR

Posts: 11

November 10, 2008

In past I have noticed that studying mitochondrial oxidative phosphorylation using Plexiglas incubation chamber can cause problem when respiratory chain inhibitors/uncouplers are used. They may stick to the plastic and leach in the next run, giving falls data. Also, I have noticed that when one uses glass fiber filters lot of calcium can leach in the filtrate changing the calcium concentration of the filtrate. Always keep your eyes and mind open while doing the experiments.
Avatar of: anonymous poster

anonymous poster

Posts: 4

November 10, 2008

For years my mentor's lab had used a specific brand of transwells for culturing uterine epithelial cells. Abruptly rat uterine, but not mouse or human uterine cells, failed to grow in these transwells. It took six months of reagent tests to determine the source of the problem. When the manufacturer was advised of the problem, nothing but excuses was offered. Needless to say, we changed brands and since have been able to grow the cells required for our experiments. \n\nIn the process of testing we also found that some proteins would cling to the side walls of certain plastic culture containers. \n\nA major concern is that the pharmaceutical industry and labs that study toxins also use the same brands of plastics as those of us who do basic bench research. Their results are as subject to variation due to unannounced changes in plastics as is the case for the rest of us. \n\nPerhaps it's time that the plastics industry that supplies the needs of the scientific community be held to at very least a standard of continuity.\n
Avatar of: John Michie

John Michie

Posts: 1

November 18, 2008

It's been known for years (decades) that different plastic types and even different sources/manufacturers of the same plastic types can affect the plating efficiency and/or growth of various cell lines. It's also not just the chemicals leaching out that have an affect, there's also factors such as the static charge in the plasticware.
Avatar of: anonymous poster

anonymous poster

Posts: 1

November 18, 2008

Our lab has seen and documented kinase inhibition in the center of 384-well polypropylene plates, as well as inhibition at the wells closest to the mold injection points. Of course, the plate manufacturer has denied any issue with their plates to this point...!
Avatar of: Andy Holt

Andy Holt

Posts: 1

November 18, 2008

To the person who posted below regarding inhibition of kinases at the mould injection points of 384-well microplates:\n\nThis is a very interesting observation. I'm the author of the Science paper discussed in this article, and we have also regularly observed "uneven" activity of amine oxidase enzymes incubated in 96 well plates, with a clear pattern seen across plates. We initially assumed that the problem was due to uneven warming of the plates in the platereader incubator chamber, and saw reduced effects at 30C compared with 37C. However, that would also be consistent with more rapid leaching of inhibitors from the plates at the higher temperature. I'd be interested to hear more about what your group found; could you drop me an e-mail at your convenience? My e-mail address can be found on our Department's web pages (www.pmcol.ualberta.ca). Thanks.\n\nRe: replacing plastics with glass. Fair point, but as others have pointed out, glass comes with its own problems. Also, there is no glass equivalent for the disposable pipette tip, so whether we want to or not, we're obliged to use at least some disposable items made from polypropylene and related plastics.\n\nRe: cell culture problems. Again, a well-documented issue, although not one that we looked at specifically. Nevertheless, cell culture systems seem to me to be particularly susceptible to the sorts of issues we identified. There's no easy fix to that problem though; you can't use glassware for sterile cell culture.
Avatar of: anonymous poster

anonymous poster

Posts: 26

November 18, 2008

Since MAO inhibitors are often used as antidepressants, perhaps drinking from plastic containers will make us feel better about this issue until it is solved.\n\nBut, seriously, have there been any clean scientific studies of water, milk, juice, meat and wine stored in various plastics vs. glass? Before and after microwaving? It seems like a good set of papers for a tox lab with GC/MS equipment. Then followup characterization of any compounds found... inert? biologically active?\n\nBaxter Zappa
Avatar of: anonymous poster

anonymous poster

Posts: 2

November 18, 2008

I know of 1 lab that had/has problems with plastic tubes/plates that activate neutrophils.
Avatar of: Karen Vaughan

Karen Vaughan

Posts: 1

November 21, 2008

I have a colleague who was using bottled water from plastic bottles in order to ensure uniformity. Her cell cultures didn't grow properly.
Avatar of: Kenneth Roy

Kenneth Roy

Posts: 11

November 21, 2008

Dr. Holt has made a useful contribution but the last sentence in his comment roused my interest. He must be very young because those of us who are older (and retired) remember that glass was used for sterile cell cultures for decades. I acknowledge that glass is not perfect, but it has and can be used.
Avatar of: Walter Niles

Walter Niles

Posts: 1

December 30, 2008

The quaternary ammonium biocides are used to control the wall wetting by molten linear olefinic and polystyrene resins in the mold ducts. They are an old trick that HP developed for getting inks through the fine ducts of their print heads. Shearing of the polymer melt down the channel requires some control of the melt adhesion to the walls in order to get the bulk of the mass to flow into the work article instead of hanging up along the duct walls.\n\nAnyway, it may be time to insist on a new plastic with better flow properties and that doesn't contain these leachable chemicals. \n\nWhen Aurora Biosciences first fabricated the 48 x 72 well array 3456-well plates from polystyrene, cells couldn't survive in them. They were being overwhelmed by some type of leachate in the small, 2 microliter volumes, but we surmised it might be the residual metal catalysts, which are present at up to 5 mol% in the resin. This problem was overcome only by switching to cyclic olefin polymers. Their melt viscosity is balanced so as to obviate a need for dewetting additives. The resin is clean.\n \nA review for those interesed is in Assay and Drug Development Technologies 6(20). doi:10.1089/adt.2008.134\n\nIt is one of the few materials certified for in vivo implantation in humans.\n\nThe problem with COP now is the cost. There is so little use for it outside of high-density multiwell plates that only by demanding "box car" quantities for routine laboratory plasticware will economies of scale be realized.

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